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DISSERTATION
Titel der Dissertation
NOVEL ANTINEOPLASTIC PLATINUM(IV) COMPLEXES: SYNTHESIS,
CHARACTERIZATION, BIOLOGICAL INVESTIGATIONS AND STRUCTURE-
ACTIVITY RELATIONSHIPS
verfasst von
Hristo Varbanov
angestrebter akademischer Grad
Doktor der Naturwissenschaften (Dr. rer. Nat.)
Wien, 2013
Studienkennzahl lt. Studienblatt: A791 419
Dissertationsgebiet lt. Studienblatt: Chemie
Betreut von: ao. Univ.- Prof. Mag. Dr. Markus Galanski
Ph. D.-Thesis
NOVEL ANTINEOPLASTIC PLATINUM(IV) COMPLEXES:
SYNTHESIS, CHARACTERIZATION, BIOLOGICAL INVESTIGATIONS
AND STRUCTURE-ACTIVITY RELATIONSHIPS
Written by
Mag. Hristo P. Varbanov
Vienna, 2013
This PhD thesis is based on the following papers, which are presented in the
original format:
Synthesis and characterization of novel bis(carboxylato)dichloridobis(ethylamine)
platinum(IV) complexes with higher cytotoxicity than cisplatin.
H. Varbanov, S.M. Valiahdi, A.A. Legin, M.A. Jakupec, A. Roller, M. Galanski, B.K.
Keppler, Eur. J. Med. Chem., 2011, 46, 5456-5464.
Novel tetracarboxylatoplatinum(IV) complexes as carboplatin prodrugs.
H.P. Varbanov, S.M. Valiahdi, C.R. Kowol, M.A. Jakupec, M. Galanski, B.K. Keppler,
Dalton Trans., 2012, 41, 14404-14415.
Theoretical Investigations and Density Functional Theory Based Quantitative
Structure–Activity Relationships Model for Novel Cytotoxic Platinum(IV)
Complexes.
H.P. Varbanov, M.A. Jakupec, A. Roller, F. Jensen, M. Galanski, B.K. Keppler, J. Med.
Chem., 2013, 56, 330-344.
ACKNOWLEDGEMENTS
I would like to thank everybody who has supported me during the work on my PhD
thesis, especially:
- assoc. Prof. Dr Markus Galanski for the greatest supervision and support in every
situation which has occurred, the detailed NMR investigations of my compounds, all the
helpful discussions, ideas and suggestions towards my work and proofreading of all my
publications, conference abstracts and this PhD thesis; furthermore, for the opportunity to
work in a creative, not-stressful and friendly atmosphere.
- Prof. DDr. Bernhard Keppler for the opportunity to become a member of his group and
develop my interests in platinum-based medicinal chemistry.
- Alexander Roller and assoc. Prof. Dr Vladimir Arion for the measurement and
refinement of the crystal structures of various complexes.
- Anatoli Dobrov and Aliona Luganschi for measuring ESI-MS spectra.
- NMR service team (Wolfgang Kandioller, Verena Pichler, Michi Primik, Paul-Steffen
Kuhn, Melanie Schmidlehner and Sergey Abramkin) for measuring NMR spectra.
- the bio unit of our group for testing the in vitro cytotoxicity of my compounds (Seied
Mojtaba Valiahdi and Mahsa S. Adib-Razavi), FACS studies (Anton Legin) and Dr
Michael Jakupec for proofreading all of the obtained data and the biological part of my
papers.
- assoc. Prof. Dr Frank Jensen from the University of Aarhus for supervising my
computational studies and proofreading the last publication, included in this thesis.
- Elfriede Limberger, Susanne Kunerth and Lydia Koltaj for helping me with all the
administration.
- Harald Fuchs and Dr Werner Ginzinger for helping me solving all the occured
computer-associated problems.
- Dr Michael Reithofer and Dr Yulia Scaffidi-Domianello for helping me to orientate in
the Lab and in the group in the beginning.
- all my colleagues and especially the Platinum group team: Yulia, Verena, Björn, Sergey
and Jelena for the good and friendly competitive atmosphere and fruitful discussions.
- Sarah Theiner for proofereading this PhD thesis.
- Miljan, Masha and Sarah for the many interesting discusions during lunch.
- for the financial support:
Finally, I would like to thank my family and friends for supporting and believing in me,
despite my complicated character.
ABSTRACT
Platinum(II) complexes represent one of the most widely used classes of cytostatics in
anticancer chemotherapy. Their clinical effectiveness is accompanied by severe dose-
limiting side effects, intrinsic and/or acquired tumor resistance and the inconvenient and
cost intensive way of intravenous administration. Platinum(IV) complexes also possess
antitumor activity and their physicochemical and chemical properties could be utilized in
order to overcome the main drawbacks of platinum(II)-based drugs. The successful
design of platinum(IV) chemotherapeutics requires a careful examination of their
pharmacology and toxicology, the formulation of structure-activity relationships and the
development of new synthetic approaches.
Within this PhD work, novel bis-, tris- and tetrakis(carboxylato)platinum(IV) complexes,
designed as prodrugs for cis-[Pt(EtNH2)2Cl2] (cisplatin analogue with higher
lipophilicity), carboplatin and nedaplatin were synthesized. For this purpose, the
respective platinum(II) complexes were oxidized with H2O2 in aqueous media and further
carboxylated using different cyclic anhydrides (succinic, glutaric, 3-methylglutaric and
3,3-dimethylglutaric anhydride). The resulting compounds were subsequently derivatized
by activation of their free carboxylic groups with CDI (1,1’-carbonyldiimidazol),
followed by reaction with various amines or alcohols, yielding the desired amides and
esters, respectively. All complexes were fully characterized, using multinuclear (1H, 13C,
15N and 195Pt) 1D and 2D NMR spectroscopy, elemental analysis, ESI-MS, ATR-FTIR,
HPLC and exemplarily X-ray diffraction for some of the compounds. In vitro
cytotoxicity of the novel complexes was examined in four human tumor cell lines
originating from ovarian carcinoma (CH1 and SK-OV-3), colon carcinoma (SW480) and
non-small cell lung cancer (A549) by means of the MTT colorimetrical assay.
Comparative analysis of the lipophilicity, electrochemistry and rate of reduction by
ascorbic acid of the new complexes was conducted in order to better understand their
pharmacological behavior. Finally, computational studies with respect to the electronic
structure and redox properties of the investigated compounds, using DFT methods were
performed. Furthermore, QSAR models with good explanatory and predictive properties
for the cytotoxicity in the cisplatin sensitive cell line CH1 and the intrinsically cisplatin
resistant cell line SW480 were developed.
ZUSAMMENFASSUNG
Zytotoxische Platin(II) Komplexe gehören zu den meist eingesetzten Substanzklassen in
der Chemotherapie. Ihre klinische Effizienz geht jedoch mit schweren dosislimitierenden
Nebenwirkungen, intrinsischer und/oder erworbener Tumorresistenz und der
unbequemen und kostspieligen Art der intravenösen Verabreichung einher. Platin(IV)
Komplexe besitzen ebenfalls tumorhemmendes Potenzial und ihre physikochemischen
und chemischen Eigenschaften könnten daher zur Überwindung der Nachteile der
Platin(II) Wirkstoffe ausgenutzt werden. Für die erfolgreiche Entwicklung von
Platinchemotherapeutika in der Oxidationsstufe +4, bedarf es einer sorgfältigen
Untersuchung ihrer Pharmakologie und Toxikologie, der Erarbeitung von Struktur-
Aktivitätsbeziehungen und der Entwicklung neuer Synthesestrategien.
Im Rahmen dieser Doktorarbeit wurden neuartige bis-, tris- und
tetrakis(carboxylato)platin(IV) Komplexe, die als Prodrugs für cis-[Pt(EtNH2)2Cl2] (ein
Analogon zu Cisplatin, jedoch mit größerer Lipophilie), Carboplatin und Nedaplatin
konzipiert wurden, synthetisiert. Zu diesem Zweck wurden die entsprechenden Platin(II)
Komplexe mit H2O2 in wässriger Lösung oxidiert und dann mit unterschiedlichen
zyklischen Anhydriden (Bernstein-, Glutar-, 3-Methylglutar- und 3,3-
Dimethylglutaranhydrid) carboxyliert. Die erhaltenen Verbindungen wurden
anschließend durch die Aktivierung der freien Carboxylgruppen mit CDI (1,1’-
Carbonyldiimidazol) derivatisiert, gefolgt von einer Reaktion mit verschiedenen Aminen
oder Alkoholen, die die gewünschten Amide und Ester ergaben. Alle Komplexe wurden
durch multinukleare (1H, 13C, 15N und 195Pt) 1D und 2D NMR-Spektroskopie,
Elementaranalyse, ESI-MS, ATR-FTIR, HPLC und exemplarisch an einigen
Verbindungen durch Röntgendiffraktometrie vollständig charakterisiert. Die in vitro
Zytotoxizität der neuen Komplexe wurde mit Hilfe des MTT kolorimetrischen Assays in
vier menschlichen Tumorzelllinien, die von Ovarialkarzinom (CH1 und SK-OV-3),
Dickdarmkarzinom (SW480) und nichtkleinzelligen Lungenkarzinom (A549) herrühren,
untersucht. Zur besseren Einschätzung des pharmakologischen Verhaltens der neuen
Komplexe, wurde eine vergleichende Analyse in Bezug auf ihre Lipophilie,
Elektrochemie und der Geschwindigkeit der Reduktion mit Ascorbinsäure
vorgenommen.
Schließlich wurden Computerstudien, basierend auf DFT-Methoden bezüglich der
Elektronenstruktur und der Redoxeigenschaften der untersuchten Verbindungen
durchgeführt. Darüberhinaus wurden QSAR-Modelle mit guter Aussagekraft und
Prädiktivität hinsichtlich der Zytotoxizität in der Cisplatin sensitiven Zelllinie CH1 und
der intrinsisch Cisplatin resistenten Zelllinie SW480 entwickelt.
ABBREVIATIONS
Ac – acetate
BSO - buthionine sulphoximine
CBDCA - 1,1’-cyclobutandicarboxyllic acid
CE - capillary electrophoresis
CML – chronic myelogenous leukaemia
DACH – 1,2-diaminocyclohexane
DFT - density functional theory
DLT - dose-limiting toxicity
DMF – dimethylformamide
DMSO – dimethylsulfoxide
DNA - deoxyribonucleic acid
DTD - drug targeting and delivery
EDDA - 1,2-ethylendiaminediacetic acid
en – ethylenediamine
EPR - enhanced permeability and retention
ESI-MS – electrospray ionisation mass spectrometry
FDA – food and drug administration
GIT - gastro-intestinal tract
GSH – glutathione
GST - glutathione-S-transferase
HCC - hepatocellular carcinoma
HSA – human serum albumin
ICP-MS - inductively coupled plasma mass spectrometry
IR – infrared (spectroscopy)
i.v. – intravenous
LUMO – lowest unoccupied molecular orbital
MEEKC – microemulsion electrokinetic capillary chromatography
MIF - molecular interactions field
MMR - mismatch repair
MW – molecular weight
NADH – nicotineamide adenine dinucleotide
NCI – national cancer institute
NER - nucleotide-excision repair
NMR – nuclear magnetic resonance
NSCLC – non- small cell lung cancer
OCT – organic cation transporter
p.o. – per os
PSA - polar surface area
QM - quantum mechanics
QSAR – quantitative structure activity relationships
QSPR - quantitative structure-properties relationships
ROS – reactive oxygen species
RP-HPLC – reverse phase high-performance liquid chromatography
RT – room temperature
SAR – structure-activity relationships
SCLC – small cell lung cancer
SEC – size exclusion chromatography
STAT - signal transducer and activator of transcription
TS – transition state
XANES - X-ray absorption near edge spectroscopy
TABLE OF CONTENTS
I. INTRODUCTION 3
1. Cancer, definitions and social significance 3
1.1. Treatment of cancer 4
1.1.1 Anticancer chemotherapeutic agents 4
2. Platinum-based therapy 7
2.1. Cisplatin 8
2.2. Carboplatin 9
2.3. Oxaliplatin 10
2.4. Platinum-based drugs with regional approval 10
2.5. Platinum-based cytostatics - pharmacology and structure-activity 12
relationships (SAR)
2.5.1 Administration and fate in the blood circulation 14
2.5.2 Cellular uptake 16
2.5.3 Intracellular activation 17
2.5.4 Interactions with DNA 19
2.5.5 Deactivation of Pt(II) drugs. Resistance 20
2.6. Drawbacks of existing platinum-based therapy. Perspectives 22
3. Platinum(IV) complexes as an alternative 28
3.1. Pt(IV) complexes in clinical trials 31
3.1.1 Ormaplatin (Tetraplatin) 31
3.1.2 Iproplatin 32
3.1.3 Satraplatin 32
3.2. Proposed mechanism of action and SAR 33
3.2.1 all trans platinum(IV) complexes 35
3.3. Physicochemical properties of interest and their control 36
3.3.1 Solubility and lipophilicity 36
3.3.2 Redox behavior 39
3.3.2.1 Participation of platinum(IV) complexes in non-redox reactions, 45
which might be of clinical relevance
3.4. Synthetic approaches in Pt(IV)-based medicinal chemistry 46
3.4.1 Using simple Pt(IV) compounds as starting materials 46
3.4.2 Oxidation with Cl2 46
3.4.3 Oxidation with other halogens 47
3.4.4 Oxidation with peroxides 47
3.4.5 Oxidation with other oxidizing agents 49
3.4.6 Further derivatization of trans-chlorido and trans-
hydroxidoplatinum(IV) complexes 49
4. References 57
II. RESULTS 69 1. Synthesis and characterization of novel bis(carboxylato) 71
dichloridobis(ethylamine) platinum(IV) complexes with higher
cytotoxicity than cisplatin
2. Novel tetracarboxylatoplatinum(IV) complexes as carboplatin prodrugs 83
3. Theoretical Investigations and Density Functional Theory Based 105
Quantitative Structure–Activity Relationships Model for Novel
Cytotoxic Platinum(IV) Complexes.
III. CONCLUSIONS 145
IV. CURRICULUM VITAE 149
I. INTRODUCTION
1. Cancer, definitions and social significance
Cancer is one of the most spread and life-costing diseases in the modern society. Based
on the GLOBOCAN 2008 estimates, about 12.7 million cancer cases and 7.6 million
cancer deaths are anticipated to have occurred in 2008.1 With more than 3 million new
cases and 1.7 million deaths (20% of the mortality in Europe) each year, cancer is the
most prominent cause of death and morbidity in Europe after cardiovascular diseases.2
Cancer is a term for a group of diseases, characterized by uncontrolled division of
abnormal (‘immortal’) cells, which have lost their ability for differentiation and are able
to invade other tissues. Localized tumors, which do not spread to other parts of the body,
are called benign, while these which are able to invade nearby tissues and to set up
second type of tumors (forming metastasis), malignant. There are more than 100 cancer
types (named on the base of the organ or type of tissue in which they occur), which could
be grouped in categories3, such as carcinoma (origins from the epithelial tissue), sarcoma
(origins from the connective tissue), leukemia (origins in blood-forming tissue such as
the bone marrow and causes large numbers of blood cells to be produced and enter the
bloodstream), lymphoma and myeloma (origins in the cells of the immune system),
central nervous system cancers (begin in the tissues of the brain and spinal cord).
The most frequently diagnosed neoplasm among females is breast cancer, while prostate
cancer is the most common in males. Generally, the leading cause of cancer death for
both genders in Europe is lung cancer (20%), followed by colorectal cancer (12%),
illustrated in Fig. 1.
3
Figure 1. Estimated number and percent of cancer morbidity (left) and mortality (right)
in Europe, both sexes, all ages, according to GLOBOCAN 20081
1.1. Treatment of cancer
The classical tool for treatment of cancer is based on a combination between surgery,
radiotherapy and chemotherapy, approaches complementary to each other. Other
methods for cancer treatment are bone marrow and peripheral blood stem cells
transplantation, the use of angiogenic inhibitors, biological therapy (immunomodulators
or targeted therapy; cancer vaccines and gene therapies are in clinical studies) and
photodynamic therapy.4 The described approaches, a step to personalized medicine, are
characterized with higher selectivity compared to classical radio- and chemotherapy, but
are usually used in a combination with them.
The development of successful treatment and early diagnosis has increased dramatically
the overall survival rate of cancer patients.5 (see Fig. 2)
1.1.1. Anticancer chemotherapeutic agents
There are more than 70 clinically applied antitumor agents, characterized with relatively
low selectivity, reflecting in high general toxicity and side effects. Except the toxicity to
non-malignant cells, a main problem of anticancer chemotherapy is intrinsic and/or
4
acquired resistance of the tumor to cytotoxic agents. As the clinically applied
cytotostatics have different cellular targets and mechanisms of action, they are used in
combined therapy regimes, rather than as single agents. Anticancer chemotherapy
typically implies a combination of drugs from different classes which have dissimilar
pharmacology and toxicology. In this manner, the cure effectiveness is increased and the
toxic effects are decreased.6
Figure 2. Ten years relative survival (%), adults (15-99 years), selected cancers, England
and Wales: survival trends for selected cancers 1971-20077
There are different ways of grouping anticancer drugs, based on their chemical structure,
origin, mechanism of action, cell cycle phase specificity, etc. A short classification with
examples, based on a mixed principle is shown below:6, 8
• Alkylating and metallating agents – react directly with nucleophilic groups of DNA,
cell cycle nonspecific agents, poor selectivity:
- nitrogen mustards (melphalane, chlorambucil, ifosfamide)
- nitrosoureas (dacarbazine, carmustine)
- other alkylating agents (busulfan)
5
- platinum complexes (cisplatin, carboplatin, oxaliplatin)
• Antitumor antibiotics and derivatives – DNA intercalators, generation of reactive
oxygen species (ROS), topoisomerase II poisons:
- antracyclins (dactinomycin, doxorubicin)
- synthetic analogues (mitoxantrone)
- glycopeptides (bleomycin)
• Antimetabolites – inhibit the enzymes, involved in DNA synthesis; S-phase specific
agents:
- dihydrofolate reductase inhibitors (methotrexate)
- ribonucleotide reductase inhibitor (hydroxycarbamide)
- pyrimidine antagonists (gemcitabine, 5-fluorouracil)
- purine antagonists (6-mercaptopurine, 6-thioguanine)
• Antitumor agents from plant origin and derivatives (cell cycle specific agents):
- topoisomerase inhibitors – podophyllotoxins and camptothecins
- tubulin polymerization inhibitors (Vinca alcaloids, cryptophycins)
- tubulin depolymerization inhibitors – taxanes
• Hormone-based therapies – used for hormone dependent cancers:
- glucocorticoids, estrogens, progestins and androgens (fosfestrol)
- antiandrogens (flutamide)
- antiestrogens (tamoxifen)
- aromatase inhibitors (anastrazole, letrozole)
• Inhibitors of signaling pathways - protein kinase inhibitors and others
• Other cytotoxic agents (Asparaginase, As2O3, etc.)
6
2. Platinum-based therapy
The beginning of platinum-based therapy has its origins in Rosenberg’ serendipitous
discovery of cell division inhibition in E.coli cultures, caused by the platinum species,
formed as electrolysis products during the experiments.9 Nowadays, 34 years after
clinical approval of the first metal-based cytostatic, cisplatin (Fig. 3, (1)), platinum
compounds have profound effect in anti-cancer treatment. They are part of first line
chemotherapy in twelve neoplasms (testicular cancer, ovarian cancer, bladder cancer,
small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), head and neck
cancer, esophageal cancer, thymoma, osteogenic sarcoma, cervical cancer and colorectal
cancer).10 Moreover, platinum drugs show synergism with a wide range of cytostatics
and can be successfully combined with radiotherapy or targeted agents.
Three Pt(II) complexes have been introduced in clinics worldwide (cisplatin, carboplatin,
oxaliplatin, Fig. 3) and another five have regional approval (nedaplatin, lobaplatin,
heptaplatin, miriplatin and dicycloplatin, Fig. 4).11-13
Figure 3. Platinum complexes with worldwide clinical approval: cisplatin (1),
carboplatin (2), oxaliplatin (3).
PtNH3
NH3
O
O
O
O
Pt
H2N
NH2
O
O
O
O
( R)
(R)
PtNH3
NH3
Cl
Cl
(1) (2) (3)
7
Pt
H3N
NH3
O
O
O
O
O
O
O
OH
H
Pt
O
OO
NH3
NH3
Pt
O
O
(S)
O
H2N
H2N *
*
H3C
Pt
O
O
O
H2N
H2N(R)
(R)
O
O
Pt
H2N
NH2
O
O
(R)
(R)
O
O
(4)
(7)(6)
(5)
(8)
O
Figure 4. Platinum complexes with regional-limited clinical application: nedaplatin (4),
heptaplatin (5), dicycloplatin (6), structure taken from ref. 25, lobaplatin (7) and
miriplatin (8).
2.1. Cisplatin, (SP-4-2)-diamminedichloridoplatinum(II)
Cisplatin is the first (FDA approval in 1978) and the most used platinum-based
cytostatic; it is applied in 32 of 78 anticancer treatment regimes as a single agents or in a
combination with a wide range of other drugs.11 It has revolutionized therapy of testicular
cancer, increasing the cure rate from 5-10 % to over 90%.10 (see Fig. 5)
Treatment with cisplatin is accompanied by severe side effects such as nephrotoxicity
(dose-limiting toxicity (DLT)), neuropathy, ototoxicity, myelosuppression (reduction in
bone marrow activity), nausea and vomiting (cisplatin is the most emetogenic cytostatic).
In order to reduce the main side effects, saline hyperhydration before and after treatment,
together with diuretics (manitol) and antiemetic drugs (usually 5-HT3 antagonists) are
8
used.14 Cisplatin is administered i.v. as bolus injections or infusions in NaCl containing
solutions in order to suppress aquation. It binds covalently and irreversibly to plasma
proteins (more than 90%).15 Cisplatin is distributed in different organs and tissues with
highest concentrations in liver, prostate, kidney, ovaries; concentrations in tumors are
generally lower, than in the organ where the tumor is located. The major route of
elimination is renal excretion (first as a native product, then as metabolites). However,
platinum can be found in tissues more than 180 days after administration.16
2%6%8%
13%15%
22%34%36%
43%45%
48%52%
61%68%
76%79%
83%90%
3%6%7%
12%13%
24%38%
45%45%46%
51%61%
67%71%
78%84%
95%
PancreasLung
OesophagusStomach
BrainMultiple myeloma
OvaryLeukaemia
KidneyColon
Rectum NHL
BladderCervix UterusBreast
Hodgkin's lymphomaMelanoma
PancreasLung
OesophagusStomach
BrainMultiple myeloma
LeukaemiaKidney
RectumColon
NHLProstate
LarynxBladder
MelanomaHodgkin's lymphoma
Testis
Wom
enM
en
Five-year relative survival
10-50% survival: 29% of cases
diagnosed
More than 50% survival:
38% of cases diagnosed
less than 10% survival: 24% of cases diagnosed
More than 50% survival:
50% of cases diagnosed
10-50% survival: 27% of cases diagnosed
less than 10% survival: 15% of cases diagnosed
Figure 5. Relative five-year survival estimates based on survival probabilities observed
during 2000-2001, by sex and site, England and Wales.7
2.2. Carboplatin, (SP-4-2)-diammine(1,1-cyclobutanedicarboxylato)platinum(II)
Carboplatin is the second platinum-based cytostatic with international marketing
approval. It shows the same spectrum of activity as cisplatin, but exhibits lower toxicity
and therefore higher dosage regimes are possible. Carboplatin is a drug of choice for
treatment of advanced ovarian carcinoma.17 It is usually administered as a rapid
intravenous injection (in chloride-free solutions) or as infusion. Carboplatin has much
milder side effects in comparison with cisplatin with myelosuppresion as DLT.14
9
Similarly to cisplatin, it binds covalently and irreversibly to plasma proteins, but to a
much lower extent (under 30%). The major route of elimination is renal excretion.18
2.3. Oxaliplatin, (SP-4-2)-((1R,2R)-1,2-diaminocyclohexane)oxalato)platinum(II)
Oxaliplatin, the last platinum-based cytostatic approved for worldwide use, is the first
one showing activity against cisplatin-resistant tumors.11 It is used in adjuvant and
especially palliative therapy of metastatic colorectal cancer in combination with 5-
fluorouracil and folinic acid. Oxaliplatin is administered i.v. as short infusions. It is less
nephrotoxic and emetic than cisplatin and myelosuppression is not common; neuropathy
is the dose-limiting toxicity.14
2.4. Platinum-based drugs with regional approval
• Nedaplatin, (SP-4-3)-diammine(glycolato)platinum(II), Fig. 4, (4) was approved
for clinical use in Japan in 2005.19 Since then, it has been used in therapies of head and
neck cancer, NSCLC and SCLC. Nedaplatin is a second generation platinum-based
anticancer drug, analogue of carboplatin, whereas the CBDCA (1,1’-
cyclobutandicarboxyllic acid) ligand is exchanged with glycolate. It has the same
spectrum of activity as cisplatin and carboplatin. Nedaplatin has shown anticancer
activity comparable with that of cisplatin, but with lower nephro- and gastrointestinal
toxicity than both cisplatin and carboplatin. Analogously to carboplatin,
myelosuppression is the DLT, expressed as thrombocytopenia and neutropenia.11, 19
• Lobaplatin, (SP-4-3)-(1,2-cyclobutanedimethanamine)(L-lactato)platinum(II),
Fig.4, (7) is a third generation platinum complex which was approved in China for
treatment of chronic myelogenous leukaemia (CML) and inoperable, metastatic breast
10
and small cell lung cancer.20 It has shown less and milder side effects than cisplatin with
thrombocytopenia as DLT. Lobaplatin is a 50%/50% mixture of two diasteromers,
featuring S,S and R,R configuration of the carrier ligand.21
• Heptaplatin, (SP-4-2)-(malonato)((4R,5R)-2-(1-methylethyl)-1,3-dioxolane-4,5-
dimethanamine)platinum(II), Fig. 4, (5) was approved in South Korea for treatment of
gastric cancer, being their first domestically developed drug.22 It shows high stability in
solution, lower and milder adverse effects and cause less emesis compared with cisplatin.
DLTs are nephrotoxicity, hepatotoxicity and myelosuppression.11
• Miriplatin, (SP-4-2)-((1R,2R)-cyclohexane-1,2-diamine)bis(tetradecanoato)-
platinum(II), Fig. 4, (8) is a new lipophilic platinum-based anticancer agent, designed
specially for the intra-arterial treatment of hepatocellular carcinoma (HCC). It was
approved in Japan in 2009 for lipiodolization of HCC and it has been marketed since
2010.12, 23 The drug is administered as an iodinated poppy seed oil suspension through a
catheter inserted into the hepatic artery. 24
• Dicycloplatin, (Fig. 4, (6)) is a supramolecule, composed of carboplatin and
CBDCA, which are linked via strong hydrogen bonds.25 It was designed in order to
overcome the solubility and stability problems of classical platinum-based therapeutics.
Dicycloplatin was approved by the Chinese FDA in 2012 for prostate cancer
chemotherapy. Preclinical and clinical studies have shown superior activity and lower
adverse effects of dicycloplatin, in comparison to carboplatin.13, 26
11
2.5. Platinum-based cytostatics - pharmacology and structure-activity relationships
(SAR)
All platinum compounds in clinical use (Fig. 3 and Fig. 4) are neutral square-planar
platinum(II) complexes with cis geometry in accordance to the first established SAR27
after Rosenberg’s identification of cisplatin as lead structure.28 Their ligand sphere is
represented by two (or one bidentate) primary am(m)ines, called carrier groups and two
chlorides or a chelating carboxylate, called leaving groups. The generally accepted
mechanism of action includes intracellular activation by aquation (displacment of the
leaving group(s) by water) and subsequent covalent binding to DNA, forming DNA
adducts.29
The first step in platinum-based drug research after cisplatin’s clinical approval in 1978
was the development of less toxic analogues that retained antitumor activity. This effect
was achieved by replacement of the two chloride leaving groups in cisplatin with more
stable chelating carboxylates (second generation of platinum drugs). Exchange of the
carrier ligands (NH3) with bidentate amineligands, resulted in the third generation of
platinum-based medicines, showing activity against tumors, resistant to cisplatin (see
Table 1).
In general, the leaving groups of platinum-based drugs are responsible for their
pharmacokinetics, while the carrier ligands (which do not exchange during aquation)
determine the pharmacodynamic characteristics. Which physicochemical properties are
relevant for the pharmacology of a platinum-based cytostatic and how they are defined
from the ligands is schematized in Fig. 6.
12
Table 1. Generations of platinum-based drugs
generation carrier ligand(s) leaving group(s) examples
I NH3 Cl cisplatin
II NH3 CBDCA,
glycolate
carboplatin,
nedaplatin
III
1R,2R-DACH, 1,2-cyclobutanedime-
thanamine, 4R,5R-2-(1-methylethyl)-
1,3-dioxolane-4,5-dimethanamine
oxalate, L-lactate,
malonate
oxaliplatin,
lobaplatin,
heptaplatin
Figure 6. Relation between the ligands, physicochemical, pharmacokinetic and
pharmacodynamic properties.
The way from the administration of a platinum drug to its final target (DNA) is
schematized in Fig. 7.
13
Figure 7. Scheme of the way of a Pt(II) drug from the administration to its target
2.5.1 Administration and fate in the blood circulation
All Pt(II) complexes in clinical use are administered parenterally, because of their poor
oral bioavailability and instability in the gastro-intestinal tract (GIT).14
Important physicochemical properties on this stage are the solubility (higher water
solubility is advantageous due to i.v. administration) and stability (pharmaceutical and in
the blood circulation). Increasing water solubility and stability in aqueous solution and
blood serum is commonly achieved by replacing the chlorido leaving groups with
chelating carboxylates30 (II and III generation complexes). In the case of miriplatin,
monodentate coordinated long chained carboxylic acids are used as leaving groups,
because of the specific way of administration, where not water solubility but high
hydrophobicity is required.12 More rarely, modification of the carrier ligands could be
also used as an approach for increasing water solubility (the development of
heptaplatin).31 Another technique was applied in the development of dicycloplatin, where
increased solubility and stability in aqueous solution of carboplatin was achieved by
14
using the supramolecular concept.25 Recently, self-association of carboplatin in water
which stabilizes its solutions has also been reported.32
In the blood circulation platinum complexes bind covalently and irreversibly to plasma
proteins, but in different extent (e.g. cisplatin more than 90%, carboplatin less than 30%,
clinical data).15 Their reactivity in plasma is in general related with the strength of
leaving groups coordination. Complexes with labile leaving groups (such as H2O) are
highly toxic (high reactivity in the blood, leading to deactivation and high general
toxicity); contrary very stable complexes with inert leaving groups (SCN-, CN-) are not
active (inert intracellularly).30 Carboxylates and the more reactive chlorides have
intermediate binding strength to platinum and represent the usual leaving groups in
platinum(II)-based cytostatics. In the last decades, a couple of studies dealing with
interactions between clinical used platinum complexes and serum proteins were
performed using several analytical approaches (SEC-ICP-MS, RP-HPLC-ICP-MS, RP-
HPLC, ESI-MS, CE, NMR).33 In general, cisplatin and oxaliplatin have shown higher
affinity and faster rate of binding to the investigated plasma proteins in comparison to
carboplatin.34 However, conflicting data can be found, concerning the kinetic of binding
and the preferential binding sites.35
Despite that, the protein-free form of platinum complexes represents their active
concentration in the blood stream, protein binding could also be a tool for their selective
accumulation in tumor cells. Human serum albumin (HSA) can serve as a drug carrier
(due to the EPR (enhanced permeability and retention) effect) for different anticancer
drugs, including platinum complexes.36, 37
15
2.5.2 Cellular uptake
Cellular uptake of platinum drugs can be realized by passive, active and facilitated
transport mechanisms. For many years passive diffusion was supposed to be the main
cell entry pathway for platinum complexes.38 This consideration was supported by the
early observed linear dependence between cellular accumulation and the concentration
gradient of the applied platinum drug up to 1 mM concentrations. In addition, no
competitive inhibition between cisplatin and structural analogues was detected. As the
clinically applied platinum drugs are relatively stable in the bloodstream, they can go
through cell membranes as neutral molecules with relatively low molecular weight,
despite their hydrophilic character.39 Nevertheless, exponential relationship between
platinum uptake and lipophilicity (as the meaning of log Po/w) of platinum complexes has
been found.40
Later experiments indicated that active transport mechanisms, using carriers/channels as
well as various endocytic routes can also play important role in platinum cell uptake.
Copper transporters were demonstrated to be involved in the influx, intracellular
transport and efflux of platinum drugs. The significance of copper uptake transporter
(CTR1) for the cellular accumulation of cisplatin and its analogues can be seen from the
observed correlation between platinum drugs resistance and low expression of CTR1 in
different cancer cell lines. Furthermore, its significance was confirmed by treating yeast
and mammalian cells, in which the CTR1 gene was knocked-out, with platinum-based
cytostatics.41 Moreover, other experiments demonstrated that CTR1 is structurally
discriminative and its role in cisplatin and carboplatin accumulation differs from that of
oxaliplatin and satraplatin.39
16
The possibility to sensitize resistant tumors to platinum-based therapy by copper-
lowering agents such as D-penicillamide and trientine (expression of hCTR1 is up-
regulated under copper depleted conditions) was recently shown in vitro and in vivo in
animal tumor models. The last findings encouraged a phase I clinical trial using trientine
in sensitizing carboplatin treatment.42, 43
2.5.3 Intracellular activation
Due to the significant difference in Cl- concentrations extra- (~100 mM) and
intracellularly (3-20 mM), cisplatin undergoes activation via aquation in the cytosol,
while one or both of the chloride ions are exchanged with water molecules.38
Extracellularly, the aquation process is suppressed due to the high concentration of Cl-;
calculations showed that in such conditions 68% of the complex remains in its original
form while the rest is represented by neutral chloridohydroxido species.44 In the case of
carboplatin, which stability against hydrolysis is higher due to coordinated chelating
CBDCA and self-association processes in solution, intracellular activation differs from
that of cisplatin. Reaction with carbonates and other nucleophiles most likely play a role
for its activation in the cell, while chlorides and phosphates seem to have minor
importance.32 Theoretical and experimental studies demonstrated that the rate limiting
step in the activation of carboplatin, nedaplatin and oxaliplatin is the first aquation
process (accompanied by chelate ring opening), contrary to cisplatin where the second
hydration is rate determining.45 Consequently, the fully hydrolyzed forms of second and
third generation platinum drugs and the monoaquated form of cisplatin are suggested to
be the main products reacting with DNA. The calculations have also indicated, that in
17
acidic conditions carboplatin, nedaplatin and oxaliplatin are supposed to reach DNA in
their monoaquated form.46, 47
Another study showed that the leaving group of oxaliplatin (oxalate) is less stable
coordinated than that of carboplatin (CBDCA) and faster activation/deactivation
reactions could be expected. 48
Possible intracellular metabolites of cisplatin and carboplatin formed during the
activation processes are shown in Fig. 8.
Figure 8. Main products of intracellular activation of first and second generation
platinum drugs.
Further steps after the activation are interactions with reactive cellular components such
as proteins, membrane phospholipids, RNA, DNA, glutathione, etc., responsible for the
cytotoxicity and/or for the inactivation of platinum complexes. 38
18
2.5.4 Interactions with DNA
In order to reach their primary target (DNA), activated forms of platinum complexes
should pass through various cytosolic components and enter the nucleus, where they
react mainly with DNA, due to its high concentration there.38
There are different hypotheses, explaining why platinum drugs bind to nucleotide bases
(N-donor ligands) in the presence of cellular components like gluthatione and methionine
(S-donor ligands), with which they can form more stable platinum adducts. One of them
postulated kinetic control, a consequence of slow ligand exchange at platinum.38 Others
proposed the involvement of metallochaperons such as ATOX1 capable of delivering
platinum drugs to the nucleus.41 Nevertheless it was estimated that not more than 1% of
administered platinum ends up to its final target, DNA.49
DNA platination occurs mainly through binding to the N7 atom of guanine or adenine
bases. Platinum(II) complexes with cis configuration (all drugs in clinical use) form
predominantly bifunctional intrastrand cross-links. Nevertheless, interstrand and
monofunctional adducts were also found in small percentage. The major adducts found
for platinum-based drugs are: 65% intrastrand GG, 25% intrastrand AG, 5-10%
intrastrand GNG and 1-3% interstrand adduct formations.38 However, significant
conformational differences of the GG adducts formed with cisplatin and carboplatin and
that with oxaliplatin were observed.50 In addition, Brabec et al. showed that cisplatin and
oxaliplatin form different interstrand cross-link adducts with DNA. These differences,
caused by the exchange of ammonia with the bidentato DACH ligand, are one of the
supposed reasons for the different spectrum of activity of oxaliplatin and II generation
platinum drugs. Furthermore, the stereochemistry of the carrier ligand has a significant
influence on the character of DNA interactions and the respective cytotoxic activity.51
19
There is no clear agreement in the field which platinum-DNA adducts have the most
significant biological effect. The formed DNA adducts provoke distortions in DNA,
including unwinding and bending, which are recognized by several proteins. As a
consequence, cellular processes like replication and transcription can be inhibited, DNA-
repair mechanisms will be activated, signal-transduction pathways, controlling growth,
differentiation and stress responses will be affected, etc. The final result will be either
cell death (most likely apoptotic) or cell survival, when the DNA lesions were
successfully repaired.29 What proteins are involved in the effects caused by platinum-
DNA interactions is an object of continuous investigations.38, 52
2.5.5 Deactivation of Pt(II) drugs. Resistance
Inactivation of platinum complexes, a main reason for platinum-drug resistance, can
occur during different stages from the time the drug is administered to the moment it
interacts with DNA. First, deactivation occurs in the bloodstream where platinum
complexes can bind irreversibly to biomacromolecules and consequently their active
concentration can be decreased. However, this problem can be controlled by appropriate
choice of the leaving groups. Once entering the cell, platinum complexes undergo
aquation and the formed reactive species can be inactivated by binding to various
intracellular nucleophiles and/or by increased efflux. The major cellular mechanisms
proposed for platinum drugs resistance can be summed up to reduced cellular
accumulation, cytosolic detoxification and enhanced DNA repair and tolerance.49
Gluthatione (GSH) is the main cytosolic component, claimed to be responsible for
platinum drugs deactivation. It can form different adducts with platinum complexes,
20
including total exchange of the ligands and coordination in a bidentate chelate fashion
[Pt(GS)2]. About 60% of the intracellular cisplatin was reported to react with GSH. The
observed good correlation in several cell lines between intracellular levels of gluthatione
and sensitivity to platinum drugs suggested GSH as a major reason for tumor resistance
to platinum complexes. 29,49 Recently, Gibson showed, using NMR experiments with cell
lysates from different tumors, that gluthatione may not be the major cellular target of
cisplatin.53 Furthermore, he placed the need of reconsidering the main postulates for
mechanisms of action of platinum complexes and the models we use to establish them.54
Cellular acumulation can be reduced due to decreased influx and/or increased efflux of
the drug. Efflux proteins such as copper transporters ATP7A and ATP7B have shown
effect on platinum complexes activity as involved in their export from the cells.41
Another main reason for platinum-drug resistance is the insufficient cellular uptake, due
to low expression of CTR1 (discussed in 2.5.2).
A further major mechanism of deactivation of platinum drugs and respective tumor drug
resistance is the DNA repair/removal of platinum adducts. The nucleotide-excision repair
(NER) is the main pathway known to remove platinum drugs lesions from DNA. Another
mechanism, the mismatch repair (MMR) showed importance in recognizing and
repairing Pt-DNA adducts, caused by cisplatin and carboplatin, but not by oxaliplatin.29
As the mentioned influx/efflux transporters, cellular GSH levels and DNA repair
mechanisms capacity are different in various cancer types, the different respond to
platinum based therapy of the latter is not surprising.
21
2.6 Drawbacks of existing platinum-based therapy. Perspectives
The main drawbacks of platinum-based cytostatics, which restrict their usage can be
summarized as severe dose-limiting side effects, intrinsic or/and acquired resistance and
the inconvenient and cost intensive way of administration (i.v.). In principle, these are the
main problems, associated with cisplatin, which could not find solution in the next
generations of platinum complexes in clinical use, developed during the last 30 years.
Toxic effects were decreased, by optimizing the stability/reactivity of the compounds
through changing the chloride leaving groups for chelating carboxylato ligands. The
replacement of the ammonia carrier ligands with cyclic diamines, forming five, six and
seven membered chelate rings, was a base for the development of anti-cancer agents,
showing activity in some cisplatin-resistant tumors.31, 55, 56 However, the potential for
further improvements is rather exhausted and other strategies should be employed.
Possible approaches for overcoming the main drawbacks of platinum-based therapy are
presented below:
• Combinations of platinum drugs and new molecular targeting agents,
phytochemicals or therapeutic agents, which are able to sensitize the tumor cells to the
platinum drugs, are in different stages of preclinical and clinical trials.57, 58
• Drug targeting and delivery (DTD) methods have the aim to decrease the side
effects by selective accumulation of the cytostatics in the tumor tissue. Active DTD is
based on specific interactions between the drug and elements from the tumor cells/tissues
(e.g. transporter-, antigen or receptor-based drugs). Platinum complexes, attached to
estrogens, antiestrogens, bile acids, antimetabolites, nutrients as sugars and amino acids,
peptides as well as bisphosphonates have been developed as a trial for active targeting 58,
59. Passive DTD is based on the enhanced uptake and longer retention of drug-
22
macromolecule conjugates in tumors in comparison with normal tissues, known as
enhanced permeability and retention (EPR) effect. The used macromolecules can be
modified plasma proteins, peptides, polysaccharides, dendrimers, artificial polymers,
monoclonal antibodies, nanoparticles, etc.60
The targeting unit (biovector or macromolecule) is usually bound to the active drug via a
spacer and a linker. In the case of Pt(II) complexes, both the leaving groups and the
carrier ligands can be used for its attachment and the choice should be made based on
considerations, concerning the stability of the linker and the character of the target.59
Finally, different formulations, including lipid, nanoparticle and liposomal versions have
also been explored. Lipoplatin and aroplatin (liposomal formulations of cisplatin and the
bis(neodecanoato) analogue of oxaliplatin, resp.) are currently in clinical trials (III and II
phase, resp.).11
• Non-classical platinum(II) complexes. The design of platinum compounds,
which do not follow the general formula cis-[Pt(NHR)2L2] (where NHR or (NHR)2 is
primary or secondary mono or bischelating diamine and L or L2 is chloride or (chelating
biscarboxylate) determined from Cleare and Hoechele SAR27 can be summarized in the
following approaches:
- trans complexes
The inactivity of platinum(II) complexes with trans geometry were postulated in the
general SAR drawn by Cleare and Hoeschele27, mainly based on comparisons between
transplatin and cisplatin. The controversial biological effects of the two geometric
isomers in vitro and in vivo and the inactivity of transplatin were observed and reported
by Rosenberg in parallel with the discovery of the anticancer activity of cisplatin.28
Transplatin is much more reactive than its cis analogue and therefore it is involved in
23
higher extent in side reactions with extra- and intracellular components, leading to its
deactivation and a lower amount of the complex reaching DNA. Furthermore, due to its
geometry, transplatin is not able to form 1,2-intrastrand cross links with DNA, which are
considered to be determinative for cisplatin’s anticancer activity.
In the last two decades trans complexes, named as rule-breakers, have shown significant
cytotoxic activity in different tumor cell lines, including such resistant to cisplatin. The
breakthrough was achieved by replacing one or two of the ammonia ligands in transplatin
with sterically demanding groups, which decreases the deactivation of the resulting
compound. The development of active trans complexes is of great interest because of
their potential to overcome resistance towards classical platinum-based cytostatics due to
their capability of forming completely different Pt-DNA adducts as compared to
cisplatin, resulting in a new spectrum of activity. However, there is still no mononuclear
platinum(II) complex with trans geometry in clinical trials. The most successful trans
complexes developed up to now can be classified in the following groups: with planar
heterocyclic ligands, with nonplanar heterocyclic ligands, with asymmetric aliphatic
amines, with iminoethers and with oximes.61-63
- ammine(amine) and ammine(N-heterocycle) platinum(II) complexes
Cisplatin analogues in which one of the ammonia ligands is substituted by a bulky cyclic
amine or pyridine derivative showed enhanced activity in cisplatin-resistant cell lines.
Introduction of a sterically demanding N-containing ligand hinders the axial attack to the
platinum center of intracellular detoxifying agents such as GSH which obstruct the
deactivation of the complex. Furthermore, asymmetric compounds can form Pt-DNA
adducts different from cisplatin, which are more difficult to be recognized from the DNA
repair systems. Picoplatin, (SP-4-3)-amminedichlorido(2-methylpyridine)platinum(II)
24
(Fig. 9, (9)) is the most successful example applying that strategy, where the position of
the coordinated methylpyridine provides a steric hindrance with regard to the attack of
the drug by intacellular thiols. It has demonstrated anticancer activity in cisplatin,
carboplatin and oxaliplatin resistant cell lines and tumor models in vivo.14 Furthermore,
picoplatin is the first platinum(II) complex showing oral activity. In the moment, it is in
various phase II clinical trials.11 Picoplatin has been granted orphan drug designation
(EU/3/07/502, EMEA/OD/055/07) for the treatment of small cell lung cancer.64
Another strategy based on co-administration of unsymmetric ammine(amine) complexes
and BSO (buthionine sulphoximine, an inhibitor of GSH biosynthesis) was recently
patented.65
PtN
NH3
Cl
Cl
(9)
Pt
H2N
NH3
H3N
Cl
Pt
H2N
NH3
H3N
NH2
PtCl
NH3
H3N
NH2
4+
(10)
Pt
NH2
H2N N
N
(11)
Cl2
(NO3)4
Pt
H2N
O
H2N
O
(12)
Figure 9. Representative examples of non-classical platinum(II) complexes with
antitumor activity: picoplatin (9), BBR3464 (10), PHENSS (11), pH sensitive complex
with aminoalcoholate (12).
- positively charged multinuclear platinum complexes and platinum intercalators
Cationic platinum(II) compounds can enter the cell using organic cation transportes
(OCT). Multinuclear platinum complexes have shown increased DNA binding affinity
25
and lack of cross-resistance with cisplatin. BBR3464 (Fig. 9, (10)) is a trinuclear,
positively charged (+4) platinum(II) drug, which demonstrated impressive preclinical in
vitro and in vivo results with superior activity than the clinically applied platinum
complexes in both cisplatin sensitive and resistant tumors.66 However phase II clinical
trials have shown only partial responses of the treated cancers and poor maximum
tolerated dose of the drug.11
By chelate coordination of planar heterocyclic ligands (e.g. phenantroline derivatives) to
square planar platinum(II) complexes, compounds with DNA intercalating properties can
be obtained (Fig. 9, (11)). This type of complexes inhibit DNA replication not through
forming Pt-DNA adducts, but via reversible insertion of the molecule between the bases.
Despite the promising in vitro/in vivo activity and lack of cross resistance with cisplatin
no compound of this class is close to clinical development.60
- platinum prodrugs with an acidic pH optimum
Based on acidic conditions, presented in mainly solid tumors (pH=5-6), non-cytotoxic
compounds at physiological pH, which can be selectively activated in the tumor acidic
media have been designed. Example of complexes displaying antitumor potency with an
acidic pH optimum containg bischelating O–alkyldithiocarbonato, aminoalcoholato
ligands (Fig. 9, (12)) or monochelating 1,3-dixydroyacetonoxime have been reported in
literature.58, 67
• Platinum(IV) complexes offer a variety of advantages in comparison to their
platinum(II) counterparts. In principle, the already mentioned main drawbacks of
platinum-based chemotherapy could find a solution by switching to Pt(IV) and choosing
an appropriate coordination sphere. In addition the DTD strategy could be applied easier
using platinum(IV) compounds (discussed in details in chapter 3).
26
• Nonplatinum metal complexes have also been of great interest during the last
decades as they are generally less toxic than platinum compounds and exert anticancer
activity without mimicking cisplatin‘s mode of action.68 The most promising candidates
for new metal-based drugs are complexes of Ru(III), Ga(III), Ru(II), Au(III) and Au(I)69-
71 (see Fig. 10)
Ru
Cl
Cl Cl
Cl
N
S
NH
O
-
Ru
Cl
Cl Cl
Cl
-
HN
N
NH
N
NAMI-A
NH
+HN
Na+
counterion
KP1019
KP1339
Ga
N
O N
N
O
O
KP46
Ga
O
O O
O
O
O
O
O
O
gallium maltolate
P
RuCl
ClNH2
RuCl
H2N
+
N
N
N
RAPTA-CAu
S
S
X
X
N
HN
O
O
O
X =Cl, Br
Figure 10. Metal-based cytostatics which were/are in clinical trials (upper row) and such
in preclinical development (bottom).
27
3. Platinum(IV) complexes as an alternative
It is worth mentioning, that prior to identification of cisplatin as major cytotoxic agent,
the effects of its Pt(IV) analogue ((OC-6-22)-diamminetetrachloridoplatinum(IV)) were
noticed.72, 73 Platinum(IV) complexes possess octahedral geometry with six coordinated
atoms and exhibit higher kinetic inertness in comparison with their square-planar Pt(II)
counterparts. Irreversible, two-electron reduction, accompanied by loss of two axial
ligands is more common than ligand-exchange reactions. (Table 2)
Table 2. General comparison between Pt(II) and Pt(IV) complexes.
oxidation state +2 +4
electronic configuration [Xe]4f145d8 [Xe]4f145d6
geometry of the complexes square planar octahedral
coordination number 4 6
preferred reactions ligand-exchange reduction
Platinum(IV) complexes’ physicochemical and chemical properties could be used in
overcoming main problems of platinum-based therapy in the following manner:
a) decreasing general toxicity and side effects by using the prodrug concept,
selective tumor accumulation and/or selective activation in the tumor
It is generally accepted, that Pt(IV) complexes have to be reduced in vivo to their active
Pt(II) counterparts in order to exhibit their cytotoxic activity. In the ideal case, the
compound is stable in the bloodstream and is reduced intracellularly (Pt(IV) complexes
are considered as cellular prodrugs of Pt(II) compounds).58 It can be speculated, that
28
Pt(IV) compounds would be predominantly activated (reduced) in the tumor environment
due to its hypoxic character.
DTD strategies can be successfully applied for platinum(IV) complexes. Their kinetic
inertness and the reduction with expected loss of the axial ligands give a plethora of
possibilities for attaching different biovectors or macromolecules to one or both of the
axial ligands. Advantageously, the use of special bio-degradatable spacer between the
complex and the targeting agent is not required in this case, as this role is carried out by
the axial ligands themselves. As an example, Lippard and coworkers have successfully
conjugated carbon nanotubes and gold nanoparticles to monocarboxylato Pt(IV)
complexes as prodrugs for cisplatin.74, 75
Finally, inert and non toxic platinum(IV) prodrugs which can be photoactivated in and
around the tumor are in current development. Good examples in this direction are
photoactive complexes, featuring equatorial iodido or azido ligands and hydroxide or
acetate as axial ligands (see Fig. 11 for examples).70, 76-78
Figure 11. Platinum(IV) prodrugs, inactive prior photo activation
29
b) switching to oral administration
The higher kinetic stability of Pt(IV) complexes (they can survive in the gastro-intestinal
tract, GIT) and the possibilities for easy tuning of their lipophilicity, open a way for the
design of compounds with good oral bioavilability.58 Consequently, satraplatin,
employed as hard gelatin capsules was tested in clinical trials, being the first oral
platinum-based cytostatic.14
c) overcoming platinum drug resistance
The increased number of ligands and synthetical approaches give broader possibilities
for the design of cytostatics, able to overcome some of the factors, determining the lack
of activity of platinum(II) complexes in various cancers. Additionally, therapeutically
active molecules can be coupled to the axial ligands; after reduction in the hypoxic tumor
tissue, the reactive platinum(II) species and compounds with their own anticancer
activity or/and capable of sensitizing the tumor cells to the reduced platinum complex
will be released. Exploring this strategy, Lippard et al. have attached cell-sensitizing
estradiol units to platinum(IV) compounds as prodrugs of cisplatin (Fig. 12, (17)).60, 65
The same group has developed Mitaplatin (Fig. 12, (18)), a prodrug, designed to release
cisplatin and the orphan drug dichloroacetate after in vivo reduction. This approach offers
a dual-killing mode that can only be effective in cancer cells.79 Another example is the
nitroplatinum(IV) complexes, designed to inhibit STAT (signal transducer and activator
of transcription) protein functions (Fig. 12, (20)).65 Dyson et al. have developed Pt(IV)
derivatives of cisplatin, featuring the cytosolic glutathione-S-transferase (GST) inhibitor,
etacrynic acid. The approach aimed to overcome GST related Pt-drug resistance (Fig. 12,
(19)).80
30
Figure 12. Examples of Pt(IV) complexes, bearing bioactive molecules, able to sensitize
tumor cells to platinum therapy.
3.1 Pt(IV) complexes in clinical trials
3.1.1 Ormaplatin (Tetraplatin)
(OC-6-22)-tetrachlorido(trans-1,2-cyclohexanediamine)platinum(IV) forms similar
active species compared to oxaliplatin after in vivo reduction and following aquation.
Therefore, it has shown activity in some cisplatin-resistant tumors. Tetraplatin has been
tested in six different phase I clinical trials, sponsored by NCI (USA) and cumulative
severe neurotoxicity was observed as dose limiting toxicity.11 No further clinical trials
have been reported in literature, probably due to the development of oxaliplatin, a Pt(II)
analogue with better solubility and toxicological profile.
31
Figure 13. Platinum(IV) complexes, which have been/are investigated in clinical trials:
tetraplatin (21), iproplatin (22) and satraplatin (23).
3.1.2 Iproplatin
(OC-6-33)-dichloridodihydroxidobis(isopropylamine)platinum(IV) is the most studied
platinum-based drug in clinics, which has not gained approval so far. More than 1000
patients participated in clinical trials, including five phase I, 22 Phase II and a single
Phase III trial. Iproplatin showed a mild toxicological profile with myelosuppression as
DLT and lack of activity in the majority of cancer types tested.11 As a final outcome of
the clinical trials, iproplatin has demonstrated no advantages in comparison to
carboplatin (which has gained its approval at that time) and therefore further
development was discontinued.81
3.1.3 Satraplatin
(OC-6-43)-bis(acetato)amminedichlorido(cyclohexylamine)platinum(IV) is the first oral
administered platinum-based cytostatic tested in clinical trials. It has shown activity in
both cisplatin sensitive and resistant tumor models in vitro and in vivo. In addition, in
vivo experiments with mice demonstrated no reduction of satraplatin’s therapeutic index
when given orally (in comparison with i.p. administration).14 Preclinical and phase I
32
clinical trials showed that satraplatin has non-linear pharmacokinetics and improved
absorption and tolerability can be achieved by daily x 5 schedule administration.
Leukopenia and thrombocytopenia were found as DLT. Satraplatin’s absorption is rapid
and a high amount of the complex is irreversibly bound to plasma proteins.
Biotransformation is fast and more than six new species can be found in patient’s plasma
ultrafiltrates 15 min after application, while the parent drug cannot be detected anymore.
The products of biotransformation are platinum(IV) and platinum(II) complexes, with
(SP-4-3)-amminedichlorido(cyclohexylamine)platinum(II) detected as main metabolite.82
The outcome of different phase II studies conducted, suggested a phase III clinical trial to
be performed in patients with refractory prostate cancer. The so-called SPARC study
(satraplatin and prednisone against refractory cancer versus placebo plus prednisone) was
performed in 950 patients with hormone refractory prostate cancer who had progressed
after initial chemotherapy. Despite the positive outcome of the trial, expressed as reduced
risk of cancer progression and increased progression free survival rate, the FDA has
rejected satraplatin on the basis of its non-convincing benefits in terms of overall
survival. Currently satraplatin is in various phase I, II and III clinical trials in
combination with a range of anticancer drugs.11, 14
LA-12, a satraplatin analogue featuring bulky adamantylamine instead of
cyclohexylamine, has shown promising results in preclinical studies83. A phase I clinical
trial has been conducted, but results have not been published yet.
3.2 Proposed mechanism of action and SAR
As platinum(IV) complexes are considered to be prodrugs of platinum(II) compounds,
their intracellular pharmacology includes activation and following interaction with the
33
main target, DNA.58 The main differences in both species could be found in their
pharmacokinetics.
A platinum(IV) complex, which possesses optimal anticancer drug properties, should:
- have adequate solubility, sufficient stability in the GIT and optimal lipophilicity
(log Po/w 0.5-3.5) in order to be absorbed from the intestinal lumen into the bloodstream
(when applied orally);
- be relatively stable in the bloodstream and accumulate predominantly in the tumor;
- be reduced fast enough intracellularly to the desired platinum(II) complex.
As a consequence of the activity of ((OC-6-22)-diamminetetrachloridoplatinum(IV)) and
the first postulated SAR for platinum-based drugs84, most investigated platinum(IV)
complexes have been designed as prodrugs for platinum(II) compounds in clinical use
and their analogues (e.g. oxoplatin, tetraplatin, iproplatin). They can be presented by the
following general formula: cis,cis,trans-[PtA2L2X2], where A is the carrier group, usually
am(m)ine or N-containing heterocycle, L is regularly chloride, but can also be iodide,
azide or carboxylate and X can be Cl, OH or different functionalized carboxylates. In
analogy to platinum(II) complexes, the carrier groups, which stay on the complex after
activation (reduction and aquation), are in charge for the character of the DNA adducts
formed. The other four ligands (two axial and two equatorial) are usually released after
activation with biological reducing agents (e.g. gluthatione, ascorbate, methionine) and
following ligand-exchange reactions and are therefore responsible for the
pharmacokinetics of the drug. It has been shown, that for complexes of the type
Pt(am(m)ine)2Cl2X2, X=Cl leads to fast reduction and high systemic toxicity, X=OH
reflects in very slow kinetic of activation and consequent low activity, while X=OAc
ensures compounds with optimal redox properties.58 Nevertheless, during the last years,
34
it was demonstrated, that the redox behavior and the corresponding toxicity of
platinum(IV) complexes depend on both the axial and equatorial ligands (L and X) and
that reconsidering of classical concepts is required85 (discussed in detail in chapter 3.3.2).
It should be mentioned, that in principle platinum(IV) complexes are able to form
adducts with DNA bases.86 However this process is without clinical relevance due to its
very slow kinetic.87
3.2.1 all trans platinum(IV) complexes
Platinum(IV) complexes in which not only the axial, but also the equatorial ligands are in
trans configuration are considered to have all trans geometry. JM335 ((OC-6-12)-
ammine(cyclohexylamine)dichloridodihydroxido)platinum(IV)) was the first compound
from this type, demonstrating in vitro and in vivo antitumor activity and showing a
different cross-resistance pattern to that of its cis-isomer.88 Investigation of series of
compounds, following the general formula all trans-
amine(ammine)dichloridodihydroxidoplatinum(IV) confirmed the results obtained with
JM335. Nevertheless, the observed activity against human ovarian carcinoma xenografts
was lower than that of cisplatin.89 Quiroga and coworkers have recently showed, that
complexes of the type trans,trans,trans-[Pt(amine)(amine’)Cl2(OH)2] demonstrate
slightly higher cytotoxicity in vitro in comparison to their Pt(II) counterparts.90
However, when analyzing these results, it should be taken in account that in all trans-
[PtA2Cl2(OH)2], both chlorides and hydroxides can be considered as axial ligands and
therefore easier and faster reduction in comparison to their cis,cis,trans- analogues could
be expected.
35
Martinez et al. have reported a comparison between the biological properties of trans-
[PtCl2(NH3)(4-hydroxymethylpyridine)] and all trans-[PtCl4(NH3)(4-hydroxymethyl-
pyridine)], showing four times lower activity of the Pt(IV) analogue, but its ability for an
easy activation from the reducing agents in the cell.91 Finally, all trans-platinum(IV)
complexes have shown to be promising phototherapeutics, when featuring azides and
hydroxides as leaving groups76 (see above, Fig. 11, (14) and (15)).
3.3 Physicochemical properties of interest and their control
3.3.1 Solubility and lipophilicity
Solubility and lipophilicity are important physicochemical parameters for every drug-like
molecule playing a crucial role for the way of administration and the following
pharmacokinetic processes of absorption, distribution and excretion. Orally administered
platinum-based drugs should pass different cell membranes, getting through the gut wall
in the blood supply and after distribution to the tumor tissue, entering into the cancer
cells. Too polar molecules will not pass through the fatty cell membranes of the intestinal
lumen, while too hydrophobic will dissolve in the fat globules and will be excreted with
feces. Optimal lipophilicity, which usually corresponds to log Po/w in the region of + 0.5
to 3.5, is a main requirement for good oral bioavailability. However, drug molecules
should also possess chemical and metabolic stability in order to survive the digestive
enzymes in the GIT as well as liver metabolic enzymes.8 Although aqueous solubility is
less critical for oral administration in comparison to i.v., it plays a role for the drug
absorption efficiency, as only the dissolved fraction can be absorbed in the digestive
tract.
36
Lipophilicity and water solubility are both dependent on all six ligands from the
platinum(IV) coordination sphere. Nevertheless, these physicochemical parameters can
be most conveniently tuned by modification of the axial ligands. At first, Kelland et al.
have reported that increasing the size of the axial chains (resp. the lipophilicity) in
cis,trans,cis-(alkylamine)(ammine)bis(carboxylato)dichloridoplatinum(IV) complexes
leads to increased cytotoxicity, most likely due to enhanced cellular accumulation.92
Augmented cytotoxic activity was also observed by increasing the size/lipohilicity of the
alkylamine ligands. However, changing the carrier groups can also alter the character of
the final Pt-DNA adducts formed and therefore is not a preferable approach for adjusting
pharmacokinetic properties of a platinum(IV) complex.
In order to judge the lipophilicity of a compound and to compare platinum complexes
with different set of ligands, better parameter than number of carbon atoms in the axial
chain or the molecular weight of a complex is required. In medicinal chemistry, the
partition coefficient between water and n-octanol, log Po/w as well as the RP-HPLC
derived retention index log kw are widely used and can give a relevant quantitative
estimation of the lipophilicity of a metal-based drug. Therefore, various methods for an
experimental determination of log Po/w of platinum complexes, beyond the classical
shake-flask method, including HPLC and MEEKC have been developed.93, 94 The
isocratic and extrapolated retention factors (log kw and log k30), obtained by RP-HPLC
have also proved to be informative while studying the influence of lipophilicity in series
of cytotoxic platinum(IV) complexes.95-97 Recently, QSPR (quantitative structure-
properties relationships) models able to predict lipophilicity (as log Po/w or log kw) of
Pt(IV) complexes, using QM (quantum mechanics) or MIF (molecular interactions field)
descriptors have been developed in Osella’s group.95, 98
37
A series of compounds with the general formula [Pt(en)Cl2(OCO(CH2)nCOOR)2] and
[Pt(en)Cl2(OCO(CH2)nCONHR)2] have been synthesized and investigated in our
group.99-101 A good correspondence between lipophilicity, platinum accumulation and
cytotoxicity was observed by increasing the size of terminal ester chains, while amide
derivatives showed lower activity than expected from their log Po/w values.102 As a next
step, changing the carrier amine and tuning the lipophilicity by suitable derivatization of
the axial ligands, yielded complexes with up to 17 times higher in vitro cytotoxicity,
compared to cisplatin.103 Applying the same approach for the development of highly
active tetracarboxylatoplatinum(IV) complexes as prodrugs for carboplatin was not
successful. Remarkably, increasing the lipophilicity by variation of the axial ligands
resulted not in a promising cytotoxicity, namely higher than that of the precursor,
carboplatin.96
Despite, platinum(IV) complexes’ activity showed to be dependent on their lipophilicity,
their cytotoxic potential cannot be assumed only from this single parameter.
It should be also mentioned that the frequently observed linear relationship between in
vitro determined cytotoxicity and lipophilicity in a series of analogues is often not valid
when the compounds are tested in vivo. The main reason for that discrepancy in the case
of oral administration is the low bioavailability of very lipophilic compounds due to their
dissolution in the fat globules in the intestine. Moreover, high lipophilicity usually leads
to low water solubility and a compromise between these two parameters should be found,
as demonstrated for series of analogues with the general formula
[Pt(DACH)(OCOR)4].104 Finally, compounds with very high in vitro cytotoxicity
sometimes have a small therapeutic index in vivo, due to a high general toxicity and the
low lethal dose.
38
3.3.2 Redox behavior
How easy, how fast and to which products platinum(IV) complexes will be reduced in
the body, are main issues influencing their activity and toxicity. It is crucial to understand
how to control these parameters in order to design a successful platinum(IV) drug which
will stay in its native form until the moment it will enter the tumor cell where it will be
reduced fast enough to develop its cytotoxic effects. Primary, the electrochemical derived
redox potentials have been suggested as indicators for the redox activity of platinum(IV)
complexes. Hambley et al. have reported, that for compounds with the general formula
[Pt(en)Cl2X2], the highest redox potential (-224 mV) is observed for X=Cl, the lowest (-
884 mV) for X=OH, while for X=OAc, propionate or butyrate, intermediate values
between -490 and -520 mV were measured. The determined cathodic reduction potentials
showed good correlation with the DNA binding activity of the complexes (the more
readily reduced complexes bind to DNA in higher extent).105 However, redox potentials
are thermodynamic data and parameters, corresponding to the kinetic of reduction are
required for better estimation of platinum(IV) complexes’ biological activity.
Furthermore, as PtIV�PtII reduction is an irreversible process, due to the loss of two
ligands, cyclic voltammetric measurements often suffer from good reproducibility and
big sd values are reported in experimental data. Platinum(IV) drugs can be reduced by
both extracellular and intracellular reducing agents such as gluthatione, ascorbic acid,
methionine and others. Kinetic rate constants for the reduction by ascorbic acid were
reported as estimates for the redox behavior of iproplatin and tetraplatin in vivo. Choi et
al. described for the first time the relation between cathodic redox potentials, rate
constants of reduction and cytotoxicity of a group of platinum(IV) complexes, featuring
equatorial chlorido ligands. A rough correlation between thermodynamic and kinetic
39
redox parameters have been demonstrated in the study. The authors showed, that
reduction rates and redox potentials are mainly dependent on the axial ligands and
change in the order: OH < OCOCH2R < Cl < OCOCF3. To a lower extent, but also
meaningful, reduction was dependent on the bulkiness of both equatorial and axial
ligands; easier and faster reduction occurred when a bulkier ligand, destabilizing the six-
coordinated sphere, was present. Good correlation between cytotoxicity and rate of
reduction was only observed in homologous series (where only one ligand position is
variable).106 The last finding is understandable, as changing the equatorial ligands can
affect not only the redox behavior, but also the lipophilicity and the pharmacodynamic
characteristics of the complex.
Kratochwil and Bednarski have compared complexes of the type [Pt(en)Cl2X2] and
[Pt(en)I2X2], where X=Cl, OH, OAc, OCOCF3 and OSO2CH3 and demonstrated higher
redox potentials, reduction rates and faster cancer cell growth inhibition for diiodido
derivatives compared to their dichlorido analogues. Moreover, the authors prposed that
reduction of the diiodido derivatives results in other products than [Pt(en)I2].107
Recently, Osella and coworkers have developed a QSPR model able to predict redox
potentials of platinum(IV) complexes from their polar surface area (PSA), total area,
energy of the LUMO and dipole moment.98
Using XANES (X-ray absorption near edge spectroscopy), Hambley and coworkers have
investigated the extent of cellular reduction of complexes of the type cis,cis,trans-
[Pt(NH3)2Cl2X2]. The percentage of reduced platinum(IV) in ovarian cancer cells after 2
h of incubation increased in the following manner: X= OH<OAc<Cl, corresponding to
the trend found from the redox potential measurements. However, after 24 h of
incubation all three complexes were fully reduced.108
40
The outcomes of the clinical trials of tetraplatin, iproplatin and satraplatin have
encouraged the belief that chloride ions as axial ligands lead to very fast in vivo reduction
and high general toxicity, while complexes featuring OH groups are inactive. The
intermediate redox potential, measured for complexes bearing carboxylates as axial
ligands promised optimal prodrug properties.58, 109
All correlations between thermodynamic redox potentials and rates of reduction observed
for platinum(IV) complexes (prodrugs of diam(m)inedichloridoplatinum(II) compounds)
showed not to be valid for diaminetetracarboxylatoplatinum(IV) complexes. Recently,
Gibson and Hambley reported that bis(acetato)platinum(IV) analogues of oxaliplatin are
reduced by ascorbic acid much more slowly, than their dihydroxido analogues, despite
possessing higher cathodic reduction potential (see Fig. 14).110 Meanwhile, we have
reported on a tremendous difference in the rate of reduction by ascorbic acid of
tetrakis(carboxylato)diammineplatinum(IV) complexes, prodrugs of carboplatin, and
bis(carboxylato)dichloridodiamineplatinum(IV) complexes. The divergence found in
redox kinetics correlates well with the observed considerable variance of cytotoxicity
between the series, but not with their close redox potentials.96 In order to find an
explanation for these phenomena, a careful look into the mechanism of reduction of
platinum(IV) complexes is required.
There are different mechanisms for the reductive elimination of two ligands from the
Pt(IV) coordination sphere, suggested on the base of kinetic measurements, using UV or
NMR spectroscopy and few theoretical studies.111-115 Reduction, mediated by the faster
inner sphere electron transfer is likely to occur only when a bridge can be formed
between the reducing agent and platinum through a polarized ligand. Such role can be
carried out by chloride or hydroxide, but not by am(m)ine or carboxylate (see Fig. 14).
41
Pt
H2N
NH2
O
O
O
O
OH
OH
Pt
H2N
NH2
O
O
O
O
O
O
O
O
HO
OH
HOO
O
O-
HO
HO
HOO
O
O-
X
(24) (25)
Ep = -0.48 Vt1/2 >14 h
Ep = -0.80 Vt1/2 ~ 3h
Figure 14. Inner sphere transition state (TS), formed between ascorbate and
dihydroxidoplatinum(IV) derivative of oxaliplatin, which facilitates the electron transfer
and results in a fast reduction (24) and the inability of the diacetato analogue to form
such a TS, resulting in a slow reduction through the outer sphere pathway (25); adopted
from ref. 129.
Therefore, reduction of diam(m)inotetracarboxylatoplatinum(IV) complexes, which
possess neither chloride nor hydroxide as ligands, proceeds slowly through an outer
sphere mechanism. In such cases, the rate limiting factor seems to be the electron transfer
to the platinum, not the breaking of Pt-L bonds. Such assumptions could explain the
observed lack of correlation between kinetics and thermodynamics as in electrochemical
experiments the electron transfer is rapid and the measured potentials are dependent
mainly on the bond energies.110
As the rate of reduction of platinum(IV) complexes strongly depends not only on the
complex, but also on the reducing agent and the pH, the possible bioreductants should be
cautiously examined. Ascorbate (two-electron reducing agent) and GSH (one-electron
reducing agent) have been considered as the main intracellular species, responsible for
reduction of platinum(IV) complexes, as they are both present in relatively high
concentration in cells. Nevertheless, there are many other biomolecules in the cell,
42
capable of reducing Pt(IV) complexes.85 Recently, Gibson and coauthors have monitored
the reduction of satraplatin and analogous complexes in aqueous extracts of cancer cells,
using [1H,15N] HSQC experiments. They divided the cell extracts into high and low
molecular weight (MW) fractions and showed that the low MW fraction (which contains
both ascorbate and GSH) are quite inefficient at reducing the tested complexes.
Furthermore the observed rate of reduction, by the high MW fraction was similar to that
of the whole non-fractioned extracts.116 These findings proposed high molecular mass
biomolecules as main reducing agents of platinum(IV) prodrugs, rather than ascorbic
acid and GSH.
Finally, the question which products are formed after reduction of platinum(IV)
complexes in the cell arises. It is generally accepted, that reduction is accompanied by
the loss of two axial ligands.109, 111 Recently, Gibson has shown using [1H,15N] HSQC
NMR spectroscopy that four different products of the reduction of trans,cis,cis-
bis(acetato)diam(m)inedichloridoplatinum(IV) complexes by sodium ascorbate were
obtained.117 Consistently, Guo et al. have reported different products of reduction of
organoamidobis(pyridine)platinum(IV) complexes, featuring chlorides or hydroxides as
axial ligands.118 Osella and coworkers have recently shown, that the reduction by GSH of
bis(carboxylato)platinum(IV) analogues of picoplatin also yields more products, than the
expected picoplatin.119 Recently, Natile and coworkers showed that the mechanism of
reduction of platinum(IV) compounds and the formed products depend on the
configuration of the complex, the bulk of the carrier ligands and the nature of the
reducing agent.120
A successful design of platinum(IV) prodrugs requires knowledge not only of their
intracellular redox behavior, but also of the amount of complex able to reach the tumor
43
cell in its intact form and the nature of the formed metabolites. Tetraplatin,
([Pt(DACH)Cl4], (21), Fig. 13) showed very rapid reduction in undiluted plasma under
physiological temperature with t1/2~3s and therefore no parent drug should be expected to
reach the cell.121 On the other site, iproplatin ([Pt(IPA)2Cl2(OH)2], (22), Fig. 13) is stable
in plasma for at least 48 h and reduces intracellularly.122, 123 Nevertheless, due to the slow
overall process of intracellular activation (reduction and following aquation of the
formed Pt(II) species), iproplatin did not show advantages in comparison with
carboplatin and was not approved for clinical use.11, 81 In the case of satraplatin
(bis(acetato)diam(m)inedichlorido platinum(IV) complex, (23), Fig. 13), an intermediate
rate of extra- and intracellular reduction could be expected. Sufficient amount of the
parent drug is estimated to reach the tumor cells and to be reduced there with satisfactory
rate. Contrary, no satraplatin in its native form was detectable in patients’ blood 15
minutes after oral administration. Furthermore, different Pt(II) metabolites were
observed, not only its precursor as could be expected.82 These results are consistent with
later observations for more than one intracellular product of reduction of satraplatin’s
analogues and the involvement of high MW agents as main reducing agents in vivo.116, 117
Carr et al. have shown, that haemoglobin, cytochrom C and liver microsomes can reduce
satraplatin in the presence of NADH. Based on its relatively high physiological
abundance and fast reaction with satraplatin (half-life time of 35.8 min), haemoglobin is
suggested as one of the key reducing agents for platinum(IV) prodrugs in the blood.124
For a better estimation of the in vivo anticancer activity and toxicity of Pt(IV) complexes
their in vitro cytotoxicity, together with their redox behavior in the blood and the
extracellular environment as well as interactions with proteins should be taken into
account. QSAR models for predicting the in vitro cytotoxic activity on the basis of redox
44
potentials, log Po/w values and constitutional and DFT derived descriptors were
developed in Osella’s group and by us as a first step in rationalizing platinum(IV)-based
drugs design. 125, 126
3.3.2.1 Participation of platinum(IV) complexes in non-redox reactions, which
might be of clinical relevance
Despite the relative inertness of platinum(IV) complexes to chemical reactions prior to
their reduction to the oxidation state +II, there are some cases of reactivity which can
have clinical relevance. Oxoplatin and JM149 (the dihydroxido analogue of satraplatin)
transform to their more reactive tetrachloridoplatinum(IV) analogues after short
incubation with 0.1 M HCl (mimicking the low pH of gastric acid), while this process has
an insignificant rate in the case of satraplatin.127 The last findings should be taken into
account when formulations containing dihydroxidoplatinum(IV) complexes are
developed for oral administration.
Hambley et al. have shown, that cis-[Pt(NH3)2Cl4] undergoes aquation while both axial
and equatorial chloride ligands can be exchanged. Substitution at the axial position was
observed prior to that of equatorial chlorides and a catalytic amount of cisplatin showed
to accelerate the process significantly. The dihydroxido and diacetato analogues of cis-
[Pt(NH3)2Cl4] showed a considerably slower rate of aquation without significance for the
stability of the complexes in aqueous media.128
Recently, Gibson has reported a fast hydrolysis of complexes, bearing axial
trifluoroacetate or dichloroacetate ligands, which form subsequently the respective
monohydroxido (fast) and dihydroxido (slow) species. As a consequence, complexes like
mitaplatin will release their active axial ligands (dichloroacetate) before reaching the cell.
Therefore, a more complicated metabolism is expected as not only the redox behavior of
45
the parent compound but also of its singly and doubly hydrolyzed forms and the rates of
hydrolysis have to be considered. On the other hand, bis(acetato)- and
bis(monochloroacetato)platinum(IV) complexes showed stability against hydrolysis.129
3.4 Synthetic approaches in Pt(IV)-based medicinal chemistry
3.4.1 Using simple Pt(IV) compounds as starting materials
Hazarika and Bora have reported the preparation of complexes of the type cis-[PtL2Cl4]
(in yields around 70%), whereas L is represented by different substituted imidazoles, via
mixing aqueous solutions of PtCl4 and 2 eq of the ligand.130 Formation of trans- isomers
of the type [PtL2Cl4] is favored by the reaction between H2[PtCl6] and
triazolopyrimidines in water-ethanol solutions.131
However, more convenient methods, providing further possibilities for derivatizations are
based on the synthesis of a platinum(II) complex with the desired coordination sphere, its
oxidation to Pt(IV) and following reactions, depending on the availability of functional
groups of the ligands.
3.4.2 Oxidation with Cl2
The preparation of platinum(IV) complexes featuring chlorides as axial ligands, by
oxidizing their Pt(II) counterparts with chlorine gas was described by Kauffman.132 The
process, when performed in aqueous solution proceeds via fast formation of a trans-
chloridoaquaplatinum(IV) intermediate and a free chloride ion; consequently, Cl- attacks
the intermediate and the trans-dichloridoplatinum(IV) complex is formed as a final
product (see Fig. 15). Recently, Margiotta et al. have confirmed this mechanism, when
studying the oxidative addition of chlorine to [Pt(1,4-DACH)Cl2] in different solvents by
46
means of 1H NMR. All intermediates formed in water, DMSO, DMF and acetone have
been detected and in the case of water and DMF, [Pt(1,4-DACH)Cl3(OH)] and [Pt(1,4-
DACH)Cl3(O-DMF)]Cl were isolated as pure solids and fully characterized by elemental
analysis, ESI-MS, NMR and IR measurements.133
Liu and Chen have demonstrated an alternative convenient method for the preparation of
trans-chlorido(hydroxido)platinum(IV) complexes, using water solution of NaOCl as
oxidizing and chlorinating agent.134
Figure 15. Mechanism of oxidative addition of chlorine to platinum(II) complexes
3.4.3 Oxidation with other halogens
Oxidation of cisplatin with an aqueous solution of bromine has been reported to produce
cis,trans,cis-[Pt(NH3)2Br2Cl2] in a yield over 60%.135 The successful oxidative addition
of bromine and iodine to platinum(II) complexes with bidentatly coordinated
benzoylthiourea derivatives in halogenated organic solvents have also been achieved.136
Complexes of the type [PtIVAI4] can be prepared by oxidizing [PtIIAI2] with iodine in
solution. The choice of the solvent(s) for the reaction and the further crystallization of the
products are based on their solubility.137
3.4.4 Oxidation with peroxides
One of the most common ways for the preparation of platinum(IV) complexes is the
oxidation of their platinum(II) counterparts with H2O2. The most probable reaction
47
mechanism includes an outer-sphere electron transfer and the formation of a tetragonally
distorted square-pyramidal intermediate between H2O2, the complex and a sixth
coordination site, associated with a molecule from the solvent. The final product is a
trans-hydroxido(solvent)platinum(IV) complex, where the OH group originates from
hydrogen peroxide and the other axial ligand (second OH, alcoholate or carboxylate)
from the solvent (see Fig. 16)138, 139
Figure 16. Mechanism of oxidation of Pt(II) complexes with hydrogen peroxide in
water, alcohols or acetic acid, based on ref. 139.
When the reaction is performed in aqueous media, trans-dihydroxido complexes are
formed.140-142 However, depending on the equatorial ligands, also other products can be
obtained. The oxidation of [PtII(EDDA)Cl2] (where EDDA is 1,2-ethylendiaminediacetic
acid) in aqueous H2O2, for example, leads to intramolecular esterification and formation
of [PtIV(EDDA)Cl2], where EDDA is six-fold coordinated143 (see Fig. 17).
Figure 17. Oxidation of platinum(II) complexes containing ethylenediamine-derived
ligands having carboxylic acid substituents.
48
Lee et al. have shown that hydrogen peroxide oxidation of Pt(II) complexes in alcohols
or liquid carboxylic acids (such as acetic and propionic acid) leads to the formation of
unsymmetric Pt(IV) species, bearing one hydroxide and one alcoholate or carboxylate as
axial ligands.144, 145 Gibson and Hambley have recently reported the generation of
monocarboxylato platinum(IV) complexes, using H2O2 or tert-butyl hydrogen peroxide
as oxidizing agent and oily or solid acids, solubilized in acetonitrile or THF.146 The
formation of platinum(IV) compounds, featuring only one free OH group of an axial
position broadens the possibility for synthesis of complexes with mixed axial ligands and
facilitates the coupling of large biomolecules or nanoparticles. For instance, soluble
single-walled carbon nanotubes as delivery systems were coupled via a succinic acid
spacer to cis,cis,trans-[Pt(NH3)2Cl2OH(OEt)] in the group of Lippard.75
3.4.5 Oxidation with other oxidizing agents
Oxidation of diam(m)inedichloridoplatinum(II) complexes with NO2 in the presence of
air and an anion, being capable to coordinate to the platinum centre during the oxidation
process was used for the synthesis of novel nitroplatinum(IV) complexes, designed to
inhibit STAT protein functions (see Fig. 12, (20)).65
The oxidation of Pt(II) compounds with (diacetoxyiodo)benzene in CH2Cl2, results in
bis(acetato)platinum(IV) compounds, where the two acetato groups are in cis position,
due to rearrangement of the intermediate.147
3.4.6 Further derivatization of trans-chlorido and trans-hydroxidoplatinum(IV)
complexes
a) exchange reactions, using silver salts
49
Kizu et al. have synthesized orally active platinum(IV) prodrugs of oxaliplatin, featuring
one chlorido and one carboxylato ligand, while treating [Pt(DACH)(ox)Cl2] with silver
salts of lipophilic monocarboxylic acids.148
b) electrophilic substitution
Platinum coordinated hydroxide(s) in axial position as nucleophiles can participate in
electrophilic substitution reactions with classical organic reagents such as anhydrides and
acyl chlorides. Most commonly, anhydrides of monocarboxylic acids in excess are
utilized as reactant and solvent (or by using CH2Cl2 as solvent) in order to synthesize the
corresponding carboxylato complexes.149, 150 An alternative approach, using acyl
chlorides in the presence of pyridine in acetone was reported by Galanski and Keppler.151
Recently, Wilson and Lippard have explored the reaction of oxoplatin ((OC-6-33)-
diamminedichloridodihydroxidoplatinum(IV)) with different alkyl and aryl isocyanates
in DMF at RT. As a result, eight novel platinum(IV) complexes, featuring axial
carbamato ligands and cytotoxicities which were similar or superior than that of cisplatin,
have been synthesized.152
The main disadvantage of the mentioned methods is the impossibility for further
derivatizations. The latter would be possible when the coupled axial ligands possess free
carboxylato, aldehydo, amino or hydroxyl group. The carboxylation of platinum(IV)
complexes, featuring free OH groups in axial position, using cyclic anhydrides (succinic,
maleic and glutaric) was reported for the first time by Navarro-Ranninger and coworkers.
The generated bis(carboxylato)bis(diamine) complexes feature free carboxylic acids at
the end of the axial ligands; however, their further derivatizations have not been
reported.153 Lippard and coworkers performed the carboxylation of oxoplatin with
succinic anhydride in DMSO at 70°C with moderate yields. Amine-modified estrogens
50
have been attached to the obtained bis(succinato)platinum(IV) compounds by the use of
diisopropylcarbodiimide-4-(dimethylamino)pyridine, a common coupling reagent,
employed in peptide chemistry.154 An improved method for carboxylation of
platinum(IV) dixydoxido complexes, using cyclic anhydrides in DMF as a solvent was
developed in our group. The uncoordinated COOH groups of the prepared complexes
were further reacted with simple amines or alcoholates in the presence of 1,1’-
carbonyldiimidazole (CDI), forming the corresponding amides or esters (see Fig. 18).101
Using this approach, a plethora of novel cytotoxic bis-, tris- and
tetrakis(carboxylato)platinum(IV) complexes have been synthesized.96, 99, 100, 103, 126, 155
Yield: 85-95%
Yield: 50-98%
Yield: 20-50%
Yield: 10-35%
Figure 18. Scheme for the synthesis of novel bis-, tris- and
tetrakis(carboxylato)platinum(IV) complexes, featuring different (ester or amide)
functionality; X = CH2, C2H4, CH2CH(CH3) or CH2C(CH3)2.
51
Main disadvantages of the method are the relatively low yields and the necessity of
purification by column chromatography at the last stage, inconvenience in terms of future
industrial manufacturing. These drawbacks can be overcome by preparing a monoester of
the respective dicarboxylic acid, transforming it into an anhydride and letting the last one
react with the chosen dihydroxidoplatinum(IV) complex (see Fig. 19).i
Yield: 40Yield: 40--90%90%
Figure 19. Alternative method for the synthesis of trans-carboxylato platinum(IV)
complexes, featuring ester function.
Recently, the synthesis of cis,cis,trans-diamminedichloridobis(4-formylbenzoato)
platinum(IV) complexes and their further derivatization by means of imine ligation were
reported. The reaction between the aromatic aldehyde functionality of the complex and
excess of hydrazide- or aminooxy-functionalized substrates resulted in yields over
90%.156
The listed approaches facilitate the formation of symmetric complexes with double
functionality in axial position, when using trans- dihydroxidoplatinum(IV) compounds as
precursors. The preparation of mixed trans-carboxylato platinum(IV) complexes using
one-pot reaction between two different anhydrides and the dihydroxidoplatinum(IV)
precursor was proposed by Song, Kim and Sohn. The final compounds were isolated
i Varbanov et al., unpublished data
52
after chromatographic purification in relatively low yields (<30%).157 In an alternative
approach, suggested by the same group, monohydroxidotris(carboxylato)platinum(IV)
complexes were first isolated and than treated with anhydrides of a second carboxylic
acid in order to esterify the free axial OH ligand.158 Lippard et al. have demonstrated,
that a monohydroxidomonosuccinato complex is formed when oxoplatin reacts with
stoichiometric amount of succinic anhydride in DMSO at RT.74 Pichler et al. have
shown, that monocarboxylato platinum(IV) complexes can be obtained by reaction of
succinic anhydride with a trans-dihydroxidoplatinum(IV) precursor, bearing bulky
equatorial amine ligands such as N,N-dimethyl-ethane-1,2-diamine159.
An alternative method for unsymmetric acylation, with the aim of achieving a better
tuning of pharmacological properties of platinum(IV) complexes, was recently published
by Fei Chin et al.. In a first step, a monocarboxylated complex was generated by reaction
of the Pt(IV) precursor with benzoic acid anhydride in DMSO under high dilution in a
good yield. The free hydroxide was further derivatized with various anhydrides,
producing complexes, bearing two different axial ligands.160 Gibson and Hambley
suggested the activation of carboxylic acid with coupling agents such as DCC
(dicyclohexylcarbodiimide) or TBTU (tetramethyluronium tetrafluoroborate) prior to its
reaction with the trans-dihydroxidoplatinum(IV) precursor. The obtained
monocarboxylated complexes can be derivatized into mixed-carboxylato species
following the same synthetic strategy.146
c) nucleophilic substitution
Despite the strong nucleophilic character of Pt(IV) coordinated hydroxides, they can be
protonated at lower pH and participate in nucelophilic substitution reactions. This
strategy is employed in the synthesis of Pt(IV) compounds, featuring chloride ions as
53
axial ligands by treating their dihydoxido analogues with concentrated HCl. The
illustrated method is cheaper and more commonly used than the one described in 3.4.1
and 3.4.2.105 However, its applicability is restricted to
diam(m)inedichloridodihydroxidoplatinum(IV) complexes, as carboxylato equatorial
ligands would be also exchanged by chlorides under these conditions.
Song, Kim and Sohn have demonstrated, that compounds of the type [Pt(DACH)(OH)4]
can be carboxylated with pure acetic, propionic or butyric acid forming the respective
tris(carboxylato)monohydroxido complexes with yields over 60%. Interestingly,
regardless of the conditions (temperature and time of reaction), only three of the
hydroxides have been carboxylated, leaving one underivatized OH at axial position.
Contrary, when [Pt(DACH)(OH)4] is suspended in 0.1 M HCl, all of the OH groups are
exchanged for Cl (see Fig. 20).
Figure 20. Nucleophilic substitution of (diamine)tetrahydroxidoplatinum(IV) complexes
with carboxylic acids and with HCl.
The authors explained the observed phenomena by the different pKa values of the
aquated ligands in [Pt(DACH)(OH)4] and higher acidity of the last aqua ligand,
54
compared to that of the used carboxylic acids, but not to HCl. The presented method for
preparation of diaminetris(carboxylato)monohydroxidoplatinum(IV) complexes is much
more convenient and with higher yields formation, compared to the alternative
electrophilic substitution.161
The preparation of tris(chelating) diaminebis(dicarboxylato)platinum(IV) complexes by
dissolving [Pt(diamine)(OH)4] type compounds in concentrated aqueous solution of
dicarboxylic acids such as malonic, 3-methylmalonic, oxalic and 1,1’-
cyclobutandicarboxylic, was recently developed in our group (see Fig. 21).ii
Figure 21. Crystal structure of (OC-6-22)-ethane-1,2-diaminebis(3-
methylmalonato)platinum(IV); ORTEP diagram with ellipsoids at 60% probability.
d) derivatisation of the equatorial ligands
Galanski et al. have synthesized a diaminetetrachloridoplatinum(IV) complex featuring
ii Varbanov et al, Novel di- and trichelate am(m)inetetracarboxylatoplatinum(IV) complexes of the type [Pt(A)(R(COO)2)2], unpublished data.
55
N,N-bis(2-hydroxyethyl)ethane-1,2-diamine as carrier ligand and further derivatized it by
means of carboxylation of the peripheral hydroxyl groups with acyl chlorides. The
demonstrated approach provides the opportunity for selective conjugation of various bio-
macromolecules with the carrier ligands of platinum-based prodrugs. 162
56
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II. RESULTS
This PhD thesis is based on the following papers, which are presented in the original
format:
Synthesis and characterization of novel bis(carboxylato)dichloridobis(ethylamine)
platinum(IV) complexes with higher cytotoxicity than cisplatin.
H. Varbanov, S.M. Valiahdi, A.A. Legin, M.A. Jakupec, A. Roller, M. Galanski, B.K.
Keppler, Eur. J. Med. Chem., 2011, 46, 5456-5464.
Novel tetracarboxylatoplatinum(IV) complexes as carboplatin prodrugs.
H.P. Varbanov, S.M. Valiahdi, C.R. Kowol, M.A. Jakupec, M. Galanski, B.K. Keppler,
Dalton Trans., 2012, 41, 14404-14415.
Theoretical Investigations and Density Functional Theory Based Quantitative
Structure–Activity Relationships Model for Novel Cytotoxic Platinum(IV)
Complexes.
H.P. Varbanov, M.A. Jakupec, A. Roller, F. Jensen, M. Galanski, B.K. Keppler, J. Med.
Chem., 2013, 56, 330-344.
69
70
1. Synthesis and characterization of novel
bis(carboxylato)dichloridobis(ethylamine) platinum(IV) complexes with
higher cytotoxicity than cisplatin.
H. Varbanov, S.M. Valiahdi, A.A. Legin, M.A. Jakupec, A. Roller, M. Galanski, B.K.
Keppler, Eur. J. Med. Chem., 2011, 46, 5456-5464.
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Author's personal copy
Original article
Synthesis and characterization of novel bis(carboxylato)dichloridobis(ethylamine)platinum(IV) complexes with higher cytotoxicity than cisplatin
Hristo Varbanov, Seied M. Valiahdi, Anton A. Legin, Michael A. Jakupec, Alexander Roller,Markus Galanski*, Bernhard K. Keppler*
University of Vienna, Institute of Inorganic Chemistry, Währinger Strasse 42, A-1090 Vienna, Austria
a r t i c l e i n f o
Article history:Received 15 April 2011Received in revised form22 July 2011Accepted 4 September 2011Available online 8 September 2011
Keywords:Platinum complexesSynthesisCharacterizationLipophilicityCytotoxicity
a b s t r a c t
A series of six novel bis(carboxylato)dichloridobis(ethylamine)platinum(IV) complexes was synthesizedand characterized in detail by elemental analysis, FT-IR, ESI-MS, HPLC, multinuclear (1H, 13C, 15N, 195Pt)NMR spectroscopy and in one case by X-ray diffraction. Cytotoxic properties of the complexes wereevaluated in four human tumor cell lines originating from ovarian carcinoma (CH1 and SK-OV-3), coloncarcinoma (SW480) and non-small cell lung cancer (A549) by means of the MTT colorimetrical assay. Inaddition, their octanol/water partition coefficients (log P values) were determined. Remarkably the mostactive (and also most lipophilic) compounds, having 4-propyloxy-4-oxobutanoato and 4-(2-propyloxy)-4-oxobutanoato axial ligands, showed IC50 values down to the low nanomolar range.
� 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
Nowadays, 35 years after the serendipitous discovery of thecytotoxic potential of cis-diamminedichloridoplatinum(II) [1],more than 50% of anticancer therapy is platinum based. All plat-inum containing drugs used in the clinics are platinum(II)compounds (cisplatin, carboplatin, oxaliplatin, nedaplatin, loba-platin and heptaplatin) [2]. However, it was shown, that Pt(IV)complexes also exhibit strong cytotoxic activity and can have someadvantages in comparison to their Pt(II) analogues [3,4]. Conse-quences of the higher oxidation state are the introduction of twoextra ligands and the change from planar to octahedral geometry.These characteristics, together with their higher kinetic inertnesscompared to their platinum(II) counterparts, opens up new possi-bilities in the design of novel platinum-based drugs (easiermodulation of the pharmacokinetic properties, more opportunitiesfor targeted therapy, oral administration, etc.) [5]. Nevertheless, noPt(IV) complex has gained clinical approval up to now [2]. FourPt(IV) compounds were in clinical trials (Fig. 1): tetraplatin wasrejected after phase I because of a high general toxicity [6]; ipro-platin was abandoned after phase III clinical trials, because it didn’t
show advantages compared with carboplatin [2]; satraplatin wasrejected after the SPARC (Satraplatin and Prednisone AgainstRefractory Cancer) phase III clinical trials [7], because it didn’t showa convincing benefit in terms of overall survival [8]; currentlysatraplatin is in phase I and II clinical trials in combination regi-mens [9]; its adamantylamine analogue LA-12 has passed phase Iclinical trials.
Considering the mechanism of action of Pt(IV) complexes, it isaccepted that they act as prodrugs via activation by reduction to theirreactive Pt(II) species [10,11]. Pt(IV) based drugs would have betteractivity and lower side effects,when they are reduced primarily in thecell; contrary, an extracellular reduction would lead to deactivationand general toxicity. Consequently, the drug’s potential stronglycorrelateswith the rate of reductionwhich depends on the respectivereductionpotential [12]. Depending on the nature of the axial ligands,platinum(IV) complexes are reduced more easily in the followingorder: CF3COO> Cl> CH3COO>OH [13] It was found that complexeswith Cl� as axial ligands were reduced very fast and showed a highgeneral toxicity (tetraplatin [14]), on the other hand, complexes withaxial hydroxido ligands were not reduced fast enough in the body toexpress their antitumor activity (iproplatin [15]). In the case of axialcarboxylato ligands, an intermediate and optimal redox potential wasobserved [16], which led to promising results, obtained in preclinicaland clinical evaluation of satraplatin and its adamantylamineanalogue LA-12.
* Corresponding authors. Tel.: þ43 1 4277 52601; fax: þ43 1 4277 52680.E-mail addresses: [email protected] (M. Galanski), bernhard.
[email protected] (B.K. Keppler).
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http: / /www.elsevier .com/locate/ejmech
0223-5234/$ e see front matter � 2011 Elsevier Masson SAS. All rights reserved.doi:10.1016/j.ejmech.2011.09.006
European Journal of Medicinal Chemistry 46 (2011) 5456e5464
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Recently, a convenient way for obtaining a series of dicarbox-ylatoplatinum(IV) complexes and modulation of their physico-chemical properties such as solubility and lipophilicity wasreported by our group [17e22]. In order to broaden the knowledgewith respect to structureeactivity relationships for that type ofcompounds and to find candidates with a promising physicochem-ical and pharmacological profile, we have synthesized a series ofbis(carboxylato)dichloridobis(ethylamine)platinum(IV) complexes.The new compoundswere fully characterized by elemental analysis,ATR IR, multinuclear NMR spectroscopy, HPLC and X-ray crystal-lography in one of the cases. Their cytotoxic properties were eval-uated in four human tumor cell lines, originating from ovariancarcinoma (CH1 and SK-OV-3), colon carcinoma (SW480) and non-small cell lung cancer (A549), by means of the MTT colorimetricalassay. In addition, their octanol/water partition coefficients (log P)were determined.
2. Result and discussion
2.1. Synthesis
The new complexes were prepared according to the reactionscheme shown in Fig. 2, starting fromK2PtCl4, which was convertedto (SP-4-2)-dichloridobis(ethylamine)platinum(II) (complex 1).Oxidation of 1 to the dihydroxido complex 2 was performed inaqueous solution, using 15% hydrogen peroxide as oxidizing agent.
Subsequent carboxylation of 2 with succinic anhydride wascarried out in absolute DMF obtaining the dicarboxylato complex.The latter was used as starting material for the synthesis ofcomplexes 4e8 via activation of its free carboxylic groups with CDI(1,10-carbonyldiimidazole) in absolute DMF under argon atmo-sphere. To the imidazolide formed in situ, the respective alcoholate/alcohol mixtures or amine were added to obtain the respectiveesters (4e6) or amide (8). Purification of the crude products wasperformed with column chromatography and re-crystallization,when necessary.
When synthesizing complex 7 following the described proce-dure, a mixture of the desired diisopropylester (7) and asymmetricmethylisopropylester (7a) in a ratio of 2:1 (according to 1H NMR)was obtained. Unfortunately separation and isolation of compound7was not successful. Formation of the mixed methylisopropylesterderivative 7a was also confirmed by ESI-MS. Most likely 7a wasformed during separation via column chromatography. Apparently,reaction of 3 after CDI activationwith isopropanol is not as fast as inthe case of complexes 4e6. As a result, the monoimidazolideplatinum complex reacted with methanol, one of the constituentsof the mobile phase. By increasing the reaction time from 24 to 72 hand avoiding the use of methanol in the mobile phase for purifi-cation, we were able to obtain exclusively pure 7 in satisfactoryyield.
2.2. Spectroscopic characterization
Structures of the starting compounds (1, 2) were proven by NMRand ATR IR spectroscopy and the new complexes (3e8) were fullycharacterized by elemental analysis, one- and two-dimensional
Fig. 1. Chemical structures of anticancer Pt(IV) complexes evaluated in clinical trials.
Fig. 2. Synthesis of novel bis(carboxylato)dichloridobis(ethylamine)platinum(IV) complexes with NMR numbering scheme; i ¼ succinic anhydride/DMF, 70 deg., ii ¼ CDI/DMF, 60deg., iii ¼ RONa/ROH, RT, iv ¼ cyclopentylamine/DMF, RT.
H. Varbanov et al. / European Journal of Medicinal Chemistry 46 (2011) 5456e5464 5457
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multinuclear NMR (1H, 13C, 15N, 195Pt), ESI-MS and ATR IR spec-troscopy and in the case of 4 also by X-ray diffraction.
Configuration of the novel platinum agents can best be estab-lished with the help of one- and two-dimensional multinuclearNMR spectroscopy. 1H and 13C chemical shifts of compounds 1e8were found in the expected range, proving the supposed structureof the complexes. Correct assignment of the signals was based on1H1H COSY, 1H13C HSQC and 1H13C HMBC spectra of the complexes.In 1H NMR spectra, oxidation and subsequent derivatization canbest be judged according to the shift of the NH2 signal from5.09 ppm in the Pt(II) complex 1 to 5.96 ppm in the dihydroxidocomplex 2, and to 7.85e7.91 ppm in the dicarboxylato complexes3e8, respectively. Also indicative are the 15N resonances, where thesignal for NH2 shifts from �40.9 ppm in complex 1 toaround �21.3 ppm in compounds 3e8. Additionally, successfulderivatizations of complex 3were observed in 13C NMR spectra. Theresonance of C-6 (uncoordinated COOH) was shifted upfield uponformation of esters 4e7 or amide 8 by ca. 2 ppm. 195Pt NMR is verypowerful technique for investigating the oxidation state and thecoordination sphere of platinum complexes. 195Pt signals forcomplexes 3e8 were detected in the region between 2849 and2853 ppm, typical for compounds with cis,cis,trans-PtIVCl2N2O2coordination [23] In comparison, the platinum(II) complex 1 reso-nates at �601 ppm, more than 3000 ppm upfield to the plati-num(IV) analogues. As expected, derivatization of 3 had noinfluence on the 195Pt signal, because the changes in the moleculeare far away from the 195Pt nucleus.
Oxidation of 1 and derivatization of 2 can also be followed in theIR spectra of the complexes. A new and intense signal at 3490 cm�1,corresponding to nPtOeH can be observed in the spectrum ofcomplex 2, in comparison with that of 1. After esterification withsuccinic anhydride this signal disappeared and in complexes 3e8new strong bands in the region 1730e1630 cm�1 (nC]O) weredetected, proving the successful carboxylation. In esters 4e7, bandswith 10e25 higher reciprocal wavelengths were detected incomparison with the complex featuring free carboxylic groups (3).However, the signal around 1710 cm�1 is missing in compound 8,because of amide formation and a strong band with a shoulderaround 1639 cm�1could be observed.
ESI-MS spectra have also confirmed the identity of thecomplexes. All new compounds (3e8) were measured in thepositive as well as in the negative ion mode. In the positive ionmode, the peak assigned to [M þ Naþ]þ displayed the highestintensity, whereas in the negative ion mode the highest intensitywas detected for [M � Hþ]�. However, a peak corresponding to[M þ Cl�]�, could also be observed in the spectra of esters(complexes 4e7). The detected m/z values as well as the isotopicdistribution were in accordance with the expected chemicalstructures 3e8.
2.3. Crystal structure of complex 4
The result from the X-ray diffraction analysis of 4 is shown inFig. 3. Crystal data, data collection parameters and structure-refinement details are given in the Experimental section. Selectedbond lengths and angles are listed in Table 1. The compound crys-tallized in the triclinic centrosymmetric space group P�1. The Pt(IV)atom has an octahedral coordination geometry with two ethylamineand two chlorido ligands in the equatorial plane and two4-methoxysuccinates coordinated in axial positions. The PteN,PteCl, and PteO bond lengths (Table 1) are well comparable withthose, observed in structurally similar complexes [17,18]. Angles inthe PtCl2N2O2 octahedron were found between 86.26 and 92.69�,and between 172.70 and 178.18�, respectively. The torsion angles(PteO1eC1eO2, and PteO5eC6eO6) were found to be close to zero.
2.4. Cytotoxicity in cancer cell lines
The new compounds were tested in comparison to cisplatin infour human tumor cell lines, originating from ovarian carcinoma(CH1, SK-OV-3), colon carcinoma (SW480) and non-small cell lungcancer (A549) with the help of the colorimetric microculture MTTassay. Except for CH1 cells, these cell lines are resistant to cisplatin,showing IC50 values about one order of magnitude higher than thatin CH1 cells. The concentration-effect curves of the testedcomplexes are shown in Fig. 4 and the obtained IC50 values aresummarized in Table 2.
Expectedly, the platinum(IV) precursor 3 featuring two COOHmoieties showed the lowest cytotoxicity in all cell lines. In accor-dance with previously published data [18,19,22], lipophilicity aswell as cellular accumulation are low for such types of complexes.Conversion of 3 to the corresponding ester derivatives 4e7 hasa significant influence on antiproliferative potency. Whereascomplex 4 is as cytotoxic as cisplatin in the cisplatin-sensitive CH1cell line, complexes 5e7 are 3 to 17 times more cytotoxic thancisplatin in the same cell line. An analogous trend was alsoobserved in cisplatin-resistant A549, SW480 and SK-OV-3 cells.Parallel to an increasing lipophilicity of the ester residue (Me, Et,Pr), the IC50 values are decreasing in all cell lines, yielding clearstructureeactivity relationships. The iPr analogue 7 is similar in
Fig. 3. ORTEP diagram of 4 displaying thermal ellipsoids at 50% probability.
Table 1Selected bond lengths (Å) and bond angles (�) in complex 4.
Bond lengths (Å)PteO1 2.039 PteN2 2.068PteO5 2.039 PteCl1 2.324PteN1 2.063 PteCl2 2.309
Bond angles (deg)N1ePt1eO1 86.26 O1ePteO5 172.70N1ePteN2 92.69 N1ePteCl1 178.18N2ePteCl1 86.26 N2ePteCl2 175.68N1ePteCl2 88.72 PteO1eC1eO2 �1.87Cl1ePteCl2 92.22 PteO5eC6eO6 �0.45
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cytotoxic potency to the Pr analogue 6 in CH1 cells, but somewhatless potent in the other cell lines. In analogy to previous observa-tions [22], the cyclopentylamide derivative is equipped with a verylow antiproliferative potency, despite its high lipophilicity (seebelow).
2.5. Lipophilicity vs. cytotoxicity
As an important factor for the pharmacokinetics and the cellularaccumulation of the new complexes, the lipophilicity was deter-mined by measuring octanol/water partition coefficients (log P) bytwo different methods; log P values, obtained by RP-HPLC and bythe shake-flask method are shown in Table 3.
Complexes 4e8 have significantly higher log P values than thatreported for cisplatin (�2.59) and platinum(II) complex 1 (�1.47)[24]. However, complex 3 is also more lipophilic than its Pt(II)congener (1), but its lipophilicity is pH-dependent, due to thepresence of two underivatized carboxylic groups (lower lip-ophilicity under physiological conditions is expected). In Fig. 5,a semi-logarithmic graph plotting the cytotoxicity versus log P ofthe new complexes is shown. In case of compounds 3e7, a lineardependency between lipophilicity and cytotoxicity could beobserved e the more lipophilic a complex, the higher its
cytotoxicity. The amide complex 8 does not match this trend e
according to its lipophilicity, a higher cytotoxicity would be ex-pected. The reasons for the latter finding are yet unclear.
2.6. Induction of apoptosis and necrosis
In order to compare the capacities of inducing apoptosis andnecrosis of one representative, namely compound 7, with those ofcisplatin, growing SW480 cultures were treated with thesecompounds in various concentrations for 48 h, then double-stainedwith annexin V-FITC and propidium iodide and analyzed byfluorescence-activated cell sorting (FACS). This method allows todiscriminate necrotic (stained by propidium iodide only), earlyapoptotic (stained by annexin V-FITC) and late apoptotic (stainedby both) from viable (unstained) cells. From the dot plots (Fig. 6), itbecomes obvious that the apoptosis-inducing potency ofcompound 7 is much higher than that of cisplatin. 50 mM ofcompound 7 reduce the amount of viable cells to 50% by inductionof both apoptosis (29%) and necrosis (21%), while the sameconcentration of cisplatin has very little effect within the chosenexposure time in the intrinsically cisplatin-resistant SW480 (coloncancer) cells.
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Fig. 4. Concentration-effect curves of complexes (3e8) in CH1 (A), SK-OV-3 (B), SW480 (C) and A549 (D) obtained by the MTT assay (96 h exposure).
Table 2Cytotoxicity of novel complexes (3e8) in comparison to cisplatin in four humancancer cell lines.
Compound IC50 (mM)a
CH1 A549 SW480 SK-OV-3
3/ReCOOH 5.6 � 1.6 50 � 8 40 � 12 120 � 174/ReCOOMe 0.16 � 0.05 2.5 � 0.9 1.0 � 0.3 2.4 � 0.15/ReCOOEt 0.061 � 0.015 1.0 � 0.4 0.30 � 0.05 1.2 � 0.36/ReCOOPr 0.014 � 0.002 0.20 � 0.03 0.11 � 0.01 0.19 � 0.037/ReCOOiPr 0.0094 � 0.0012 0.78 � 0.09 0.39 � 0.07 0.49 � 0.118/ReCONHR 0.75 � 0.10 19 � 3 6.1 � 0.6 13 � 1Cisplatin 0.16 � 0.03 1.3 � 0.3 3.5 � 0.3 1.9 � 0.3
a 50% Inhibitory concentrations in CH1, A549, SW480 and SK-OV-3 cells in theMTT assay, 96 h exposure. Values are the means � standard deviations obtainedfrom three independent experiments.
Table 3Log P values for complexes 3e8, R1 and R2, estimated by RP-HPLC and the shake-flask method.
Compound Log P values, determined by RP-HPLC in differentMeOH concentrations
Log P,determinedby theshake-flaskmethod
0% 10% 20% 30% 40% 50%
R1 �1.04 �1.03 �1.00 �0.95 �0.99 �0.88 �0.813 �0.70 �0.84 �0.79 �0.64 �1.14 �0.974 0.30 0.25 0.17 0.08 0.14 �0.10 �0.125 0.90 0.87 0.83 0.73 0.78 0.85 0.646 1.38 1.41 1.44 1.49 1.47 1.48 1.447 1.30 1.33 1.36 1.41 1.37 1.398 1.11 1.14 1.17 1.23 1.20 1.25 1.21R2 1.39 1.41 1.44 1.48 1.44 1.46 1.69
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A synopsis of all data (Fig. 7) further illustrates that compound 7is a powerful apoptotic agent, which effectively causes cell death ina dose-dependent way.
3. Conclusions
Six novel bis(carboxylato)platinum(IV) complexes have beensynthesized and fully characterized. The new compounds wereinvestigated for their lipophilic properties and their in vitro cyto-toxicity in four human tumor cell lines. Remarkably, IC50 valuesdown to the nanomolar range, up to 32 times lower compared tocisplatin, were found. Whether the very high cytotoxicity is alsoaccompanied by manageable systemic toxicity in vivo will beevaluated in future work.
4. Experimental protocols
4.1. Materials and methods
All reagents and solvents were obtained from commercialsuppliers, and were used without further purification. Methanoland ethanol were dried, according to standard procedures. Forcolumn chromatography, silica gel 60 (Fluka) was used. (OC-6-33)-Dichloridobis(ethylamine)dihydroxidoplatinum(IV) (complex 2,Fig. 2) was synthesized starting from K2PtCl4 and ethylamine, usingDhara’s [25] method with some modifications. The resultingdichlorido complex 1 was oxidized with 15% H2O2.
1H, 13C, 15N, 195Pt and two-dimensional 1H1H COSY, 1H13C and1H15N HSQC, and 1H13C HMBC NMR spectra were recorded with
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Fig. 5. Semi-logarithmic plot of lipophilicity (log P determined with RP-HPLC) of complexes 3e8 vs. cytotoxicity (IC50) in CH1, A549, SW480 and SK-OV-3 cells.
Fig. 6. FACS analysis of annexin V- and PI-stained SW480 colon cancer cells. Left column: untreated control; right column: after treatment with 50 mM cisplatin/compound 7 for48 h. The upper left quadrant contains the necrotic (stained by PI only), the lower right early apoptotic (stained by annexin V-FITC only), the upper right late apoptotic (stained byboth) and the lower left quadrant the viable (unstained) fraction of cell populations.
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a Bruker Avance III 500 MHz NMR spectrometer at 500.32 (1H),125.81 (13C), 107.55 (195Pt), and 50.70 MHz (15N) in DMF-d7 atambient temperature, using the solvent residual peak for 1H and13C as internal reference. The splitting of proton resonances in the1H NMR spectra are defined as s ¼ singlet, bs ¼ broad singlet,d¼ doublet, t¼ triplet, and m¼multiplet. 15N chemical shifts werereferenced relative to external NH4Cl; whereas 195Pt chemical shiftswere referenced relative to external K2[PtCl4] (see Fig. 2 for NMRnumbering scheme).
IR spectra were obtained with a PerkineElmer 370 FT-IR 2000instrument (4000e400 cm�1) by using an ATR unit. Intensities ofreported IR bands are defined as br ¼ broad, s ¼ strong,m ¼ medium, and w ¼ weak. Electrospray ionization mass spec-trometry was carried out with a Bruker Esquire 3000 instrumentusing MeOH as solvent. Elemental analyses were performed witha PerkineElmer 2400 CHN-Elemental Analyzer by the Microana-lytical Laboratory of the University of Vienna. Analyses indicated bythe symbols of the elements or functions were within �0.4% of thetheoretical values. Purity of novel compounds was additionallyproved by analytical reversed-phase HPLC.
4.2. Synthesis
4.2.1. (OC-6-33)-Bis(3-carboxypropanoato)dichloridobis(ethylamine)-platinum(IV) (3)
Succinic anhydride (674.3 mg, 6.738 mmol) and 653 mg(1.675 mmol) of (OC-6-33)-dichloridobis(ethylamine)dihydrox-idoplatinum(IV) were suspended in 9 mL of absolute DMF and thereaction mixture was stirred at 65 �C for 40 min and then for 2more hours at room temperature. During this time, the solid
material dissolved to form a yellow-brown solution. DMF wasremoved under reduced pressure. The residue was dissolved inacetone and filtered to give a clear, yellow solution. This solutionwas concentrated under reduced pressure, and subsequentaddition of diethyl ether led to precipitation of a pale yellow solid.The precipitate was filtrated and dried in vacuo, while heating at40 �C. Yield: 398 mg (40%). Anal. C12H24Cl2N2O8Pt (C, H, N). ESI-MS: m/z 612.6 [M þ Naþ]þ, 588.7 [M � Hþ]�. 1H NMR: d ¼ 12.52(bs, 2H, COOH), 7.87 (bs, 4H, NH2), 3.10 (m, 4H, H-2), 2.75 (m, 4H,H-4), 2.68 (m, 4H, H-5), 1.44 (t, 3JH,H ¼ 7.2 Hz, 6H, H-1) ppm. 13CNMR: d ¼ 181.3 (C-3), 174.1 (C-6), 39.9 (C-2), 31.2 (C-4), 29.9 (C-5),14.3 (C-1) ppm. 15N NMR7: d ¼ �21.2 ppm. 195Pt NMR:d ¼ 2851 ppm. IR (ATR): 3241 m, 3197 br; 2977 br; 1710 s (nC]O),1642 m (nC]O); 1357 m, 1344 m; 1238 s 1216 m, 1175 m; 1081 w,1024 w (nCeN) cm�1.
4.2.2. (OC-6-33)-Dichloridobis(ethylamine)bis((4-methoxy)-4-oxobutanoato)platinum(IV) (4)
CDI (241.1 mg, 1.4869 mmol) in absolute DMF (10 mL) wasadded to a solution of 3 (433.4 mg, 0.7342 mmol) in absolute DMF(6 mL), and the mixture was heated to 60 �C. After 10 min of beingstirred, the solution was cooled to room temperature and CO2 wasremoved by flushing with argon. Sodium methanolate (a piece ofNa in 10 mL of absolute MeOH) in absolute MeOH was added andthe solution was stirred for 24 h at room temperature. Methanoland DMF were removed under reduced pressure to form a yellowoil. The crude product was purified by column chromatography(EtOAc/MeOH, 7:1) to yield a yellow solid, which was dried invacuo. Yield: 106 mg (23%). Anal. C14H28Cl2N2O8Pt (C, H, N). ESI-MS:m/z 640.8 [M þ Naþ]þ, 616.5 [M � Hþ]�, 652.8 [Mþ Cl�]�. 1H NMR:d ¼ 7.85 (bs, 4H, NH2), 3.82 (s, 6H, H-7), 3.10 (m, 4H, H-2), 2.78 (m,4H, H-4), 2.71 (m, 4H, H-5), 1.45 (t, 3JH,H ¼ 7.2 Hz, 6H, H-1) ppm. 13CNMR: d ¼ 181.0 (C-3), 173.1 (C-6), 51.2 (C-7), 39.9 (C-2), 31.1(3JC,Pt¼ 38.2 Hz, C-4), 29.8 (C-5),14.3 (3JC,Pt¼ 35.0 Hz, C-1) ppm. 15NNMR: d ¼ �21.3 ppm. 195Pt NMR: d ¼ 2853 ppm. IR (ATR): 3221 w,3188 m; 2894 br; 1729 s (nC]O), 1647 s (nC]O); 1362m 1333s; 1259s, 1198s, 1176 s; 1088 m, 1026 w (nCeN); 683 w cm�1. Crystals,suitable for X-ray data collection, were obtained after vapor diffu-sion of diethyl ether into a methanol solution of 4.
4.2.3. (OC-6-33)-Dichloridobis((4-ethoxy)-4-oxobutanoato)-bis(ethylamine)platinum(IV) (5)
The synthesis was carried out as described for 4. The crudeproduct was purified by column chromatography (EtOAc/MeOH,9:1), then recrystallized from ethyl acetate and diethyl ether toyield a pale yellow solid. The final product was dried in vacuo.Yield: 24 mg (16%). Anal. C16H32Cl2N2O8Pt (C, H, N). ESI-MS: m/z669.1 [M þ Naþ]þ, 644.3 [M � Hþ]�, 681.0 [M þ Cl�]�. 1H NMR:d ¼ 7.86 (bs, 4H, NH2), 4.26 (m, 3JH,H ¼ 7.1 Hz, 4H, H-7), 3.10 (m, 4H,H-2), 2.77 (t, 4H, H-4), 2.69 (t, 4H, H-5), 1.45 (t, 3JH,H¼ 7.2 Hz, 6H, H-1), 1.39 (t, 3JH,H ¼ 7.1 Hz, 6H, H-8) ppm. 13C NMR: d ¼ 181.0 (C-3),172.7 (C-6), 60.2 (C-7), 39.9 (C-2), 31.1 (3JC,Pt¼ 37.3 Hz, C-4), 30.0 (C-5), 14.4 (3JC,Pt ¼ 34.5 Hz, C-1), 14.0 (C-8) ppm. 15N NMR:d¼�21.3 ppm. 195Pt NMR: d¼ 2850 ppm. IR (ATR): 3180w, 3153w;2987 w; 1727 s (nC]O); 1658 m, 1631 m (nC]O); 1369 m; 1260 s,1231 s; 1163 s; 1085 w; 1039 m, 1023 s (nCeN); 857 w, 681 m cm�1.
4.2.4. (OC-6-33)-Dichloridobis(ethylamine)bis((4-propyloxy)-4-oxobutanoato)platinum(IV) (6)
The synthesis was carried out as described for 4. The crudeproduct was purified by column chromatography (EtOAc/MeOH,11:1), then recrystallized from ethyl acetate and diethyl ether toyield a pale yellow solid. The final product was dried in vacuo.Yield: 33 mg (11%). Anal. C18H36Cl2N2O8Pt (C, H, N). ESI-MS: m/z697.1 [M þ Naþ]þ, 672.9 [M � Hþ]�, 708.9 [M þ Cl�]�. 1H NMR:
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Fig. 7. Concentration-effect curves for compound 7 (A) and cisplatin (B) with regard toapoptosis and necrosis induction in SW480 cells after 48 h exposure, measured byFACS using annexin V-FITC/propidium iodide staining.
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d ¼ 7.86 (bs, 4H, NH2), 4.18 (t, 3JH,H ¼ 6.7 Hz, 4H, H-7), 3.10 (m, 4H,H-2), 2.78 (m, 4H, H-4), 2.71 (m, 4H, H-5),1.80 (m, 3JH,H¼ 7.3 Hz, 4H,H-8), 1.44 (t, 3JH,H ¼ 7.2 Hz, 6H, H-1), 1.09 (t, 3JH,H ¼ 7.4 Hz, 6H, H-9)ppm. 13C NMR: d ¼ 181.0 (C-3), 172.7 (C-6), 65.8 (C-7), 39.9 (C-2),31.1 (3JC,Pt ¼ 31.1 Hz, C-4), 29.9 (C-5), 22.0 (C-8), 14.4(3JC,Pt ¼ 34.7 Hz, C-1), 10.1 (C-9) ppm. 15N NMR: d ¼ �21.4 ppm.195Pt NMR: d ¼ 2850 ppm. IR (ATR): 3222 m, 3192 m; 2970 w; 1731s (nC]O); 1671 m, 1654 s (nC]O); 1370 m, 1326 m; 1164 s, 1088 w;684 w cm�1.
4.2.5. (OC-6-33)-Dichloridobis(ethylamine)bis((4-(2-propyloxy))-4-oxobutanoato)platinum(IV) (7)
CDI (184 mg, 1.135 mmol) in absolute DMF (6 mL) was added toa solution of 3 (325 mg, 0.551 mmol) in absolute DMF (5 mL), andthe mixture was heated to 70 �C. After 10 min of being stirred, thesolution was cooled to room temperature and CO2 was removed byflushing with argon. 12 mL of sodium 2-propanolate (a piece of Nadissolved in 2-propanol, HPLC grade) was added to the solution andheated to 40 �C. The mixture was then stirred for 72 h at roomtemperature. 2-Propanol and DMF were removed under reducedpressure to form a yellow oil. The crude product was purified bycolumn chromatography (EtOAc/2-propanol, 10:1) and thenprecipitated with Et2O and cooled to 0 �C to give an almost whitesolid, which was dried in vacuo. Yield: 110 mg (30%). Anal.C18H36Cl2N2O8Pt (C, H, N). ESI-MS: m/z 696.8 [M þ Naþ]þ, 673.4[M � Hþ]�, 708.8 [M þ Cl�]�. 1H NMR: d ¼ 7.70 (bs, 4H, NH2), 4.93(m, 3JH,H ¼ 6.3 Hz, 2H, H-7), 2.94 (m, 4H, H-2), 2.59 (m, 4H, H-4),2.49 (m, 4H, H-5), 1.27 (t, 3JH,H ¼ 7.2 Hz, 6H, H-1), 1.21 (d,3JH,H ¼ 6.3 Hz, 12H, H-8) ppm. 13C NMR: d¼ 180.9 (C-3), 172.0 (C-6),67.4 (C-7), 39.8 (C-2), 30.9 (3JC,Pt ¼ 37.5 Hz, C-4), 30.1(C-5), 21.3 (C-8), 14.2 (3JC,Pt ¼ 32.9 Hz, C-1) ppm. 15N NMR: d ¼ �21.3 ppm. 195PtNMR: d ¼ 2850 ppm. IR (ATR): 3282 m, 3206 m; 2981 w, 2937 w;1722 s, 1702 s (nC]O); 1668 s, 1637 s (nC]O); 1375 m, 1362 m; 1304s, 1265 s, 1239s; 1106 m, 1087 m cm�1.
4.2.6. (OC-6-33)-Dichloridobis((4-cyclopentylamino)-4-oxobuta-noato)bis(ethylamine)platinum(IV) (8)
CDI (106.5 mg, 0.6568 mmol) in absolute DMF (7 mL) was addedto a solution of 3 (184.5 mg, 0.3126 mmol) in absolute DMF (5 mL),and the mixture was heated to 60 �C. After 10 min of being stirred,the solutionwas cooled to room temperature and CO2was removedby flushing with argon. Cyclopentylamine (75 mL, 0.7516 mmol) in4 mL of absolute DMFwas added to the solution and stirred for 30 hin the dark at room temperature. DMF was removed under reducedpressure to form a brown oil. The crude product was purified bycolumn chromatography (EtOAc/MeOH, 4:1) and then precipitatedwith Et2O (ultrasonic) and cooled to 0 �C to give an almost whitesolid, which was filtered off, washed with Et2O and EtOAc and driedin vacuo. Yield: 64 mg (28%). Anal. C22H42N4O6PtCl2 (C, H, N). ESI-MS: m/z 747.3 [M þ Naþ]þ, 723.1 [M � Hþ]�. 1H NMR: d ¼ 7.95 (d,3JH,H ¼ 6.9 Hz, 2H, NH-amide), 7.91 (bs, 4H, NH2), 4.26 (m,3JH,H¼ 6.8 Hz, 2H, H-7), 3.13 (m, 4H, H-2), 2.68 (m, 4H, C-4), 2.56 (m,4H, H-5), 2.00 (m, 4H, H-8), 1.84 (m, 4H, H-9), 1.70 (m, 4H, H-9), 1.62(m, 4H, H-8), 1.45 (t, 3JH,H ¼ 7.1 Hz, 6H, H-1) ppm. 13C NMR:d¼ 181.8 (C-3), 171.3 (C-6), 51.0 (C-7), 40.0 (C-2), 32.7 (C-8), 32.1 (C-4), 31.8 (C-5), 23.8 (C-9),14.4 (C-1) ppm. 15N NMR: d¼ 107.7 (CONH-amide),�20.3 (NH2) ppm. 195Pt NMR: d¼ 2489 ppm. IR (ATR): 3352m, 3210 br, 3073 br; 2871 w; 1639 s (nC]O); 1535 s, 1356w,1258m,1246 w cm�1.
4.3. Crystallographic structure determination
X-ray diffraction measurement was performed on a Bruker X8APEXII CCD diffractometer. Single crystal of 4 was positioned at40 mm from the detector, and 1839 frames were measured, each
for 20 s over 1� scan width. The data were processed using SAINTsoftware [26]. Crystal data, data collection parameters, andstructure-refinement details are given in Table 4. The structureswere solved by direct methods and refined by full-matrix least-squares techniques. Non-H atoms were refined with anisotropicdisplacement parameters. H atoms were inserted in calculatedpositions and refined with a riding model. The isotropic thermalparameters were estimated to be 1.2 times the values of theequivalent isotropic thermal parameters of the atoms to whichhydrogens were bonded. Structure solution was achieved withSHELXS-97 and refinement with SHELXL-97 [27], and graphicswere produced with ORTEP-3 [28].
4.4. Determination of lipophilicity
Lipophilicity of new complexes was determined by the shake-flask method and by reversed-phased HPLC.
4.4.1. Shake-flask methodThe log P determination of compounds (4, 5, 6 and 8) was
carried out, according to the guidelines for the shake-flask method[29] with slight modifications [20]. Weighted amounts of platinumcomplexes were dissolved in HPLC-grade water, which was pre-saturated with n-octanol, and mixed by sonication for 5 min.Afterward, the solutions were centrifuged for 5 min and theconcentration of Pt was determined by ICP-MS. Weighted amountsof that solutions were mixed with the same volume of n-octanol(pre-saturated with water) and shaken for 15. After phase separa-tion, the Pt concentration in the aqueous phase was again deter-mined by ICP-MS and the partition coefficients were calculated.
The platinum content in the aqueous phase was determined byICP-MS (Agilent 7500ce, Waldbronn, Germany), equipped witha CETAC ASX-520 autosampler (Neuss, Germany), a Scott doublepass spray chamber, and aMicroMist nebulizer. For the analysis, thesamples were diluted 1:1000 with 2.5% HCl. Every sample con-tained 0.5 ppb In as internal standard (CPI International, Santa Rosa,CA, USA).
4.4.2. Reversed-phase HPLC methodHPLC analysis was performed on a Dionex Summit system
controlled by the Dionex Chromeleon 6.60 software. The
Table 4Crystallographic data for complex 4.
4
Empirical formula C14H28Cl2N4O8PtFw 618.37Space group P�1a [Å] 6.0519(3)b [Å] 13.1190(6)c [Å] 14.0840(5)a [�] 79.521(2)b [�] 83.694(2)g [�] 85.073(3)V [Å3] 1090.41(8)Z 2l [Å] 0.71073rcalcd [g cm�3] 1.883Crystal size [mm3] 0.20 � 0.13 � 0.12T [K] 100(2)m [mm�1] 6.719R1
a 0.0357wR2
b 0.0858GOFc 0.989
a R1 ¼ SjjFoj � jFcjj/SjFoj.b wR2 ¼ {S[w(Fo2 � Fc
2)2]/S[w(Fo2)2]}1/2.c GOF ¼ {S[w(Fo2 � Fc
2)2]/(n � p)}1/2, where n is the number ofreflections and p is the total number of parameters refined.
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experimental conditionswere as follows: Agilent ZORBAXBonus-RPcolumn (4.6 mm � 250 mm); 0.1% TFA water/MeOH based mobilephases; UVevis detection set up at 210 nm; temperature of thecolumn: 25 �C; flow rate: 1 mL min�1; concentration of the inves-tigated complexes: 2.5 mM, (1 mM KI as internal standard wasadded); 25 mL injection volume. The capacity factors k0 ¼ (tR� t0)/t0(tR is the retention time of the species analyzed and k0 is the reten-tion time of the unretained substance, used as a standard (here KI))of the investigated compounds were determined at differentMeOH/water ratios (from 60:40 for the most lipophilic to 10:90 forthe most hydrophilic compounds). Using the linear relationshipbetween log k’ and the percentage ofMeOH in themobile phase: logk0 ¼ log k0we %MeOH. log k0 values for all complexeswere calculatedfor 0,10, 20, 30, 40 and 50% ofMeOH in themobile phase. Complexes3e8 and for comparison (OC-6-33)-dichlorido(ethane-1,2-diamine)-bis{(4-methoxy)-4-oxobutanoato}platinum(IV) (R1) and (OC-6-33)-bis{(5-butyloxy)-5-oxo-3-methylpentanoato}dichlorido(ethane-1,2-diamine)platinum(IV) (R2) described in Ref. [22] were investi-gated. Calibration curves for different MeOH concentrations werecreated on the basis of determined log P values of 4-6, 8, R1, and R2.The equations derived from the calibration curves for differentpercentage of MeOH together with their R2 values, are shown inTable 5. From these equations log P values for all investigatedcomplexes were calculated.
4.5. Cell lines and culture conditions
CH1 (ovarian carcinoma, human) cells were donated by Lloyd R.Kelland (CRC Center for Cancer Therapeutics, Institute of CancerResearch, Sutton, U.K.). A549 (non-small cell lung cancer, human)and SW480 (colon carcinoma, human) cells were kindly providedby Brigitte Marian (Institute of Cancer Research, Department ofMedicine I, Medical University of Vienna, Austria), and SK-OV-3(ovarian carcinoma, human) cells by Evelyn Dittrich (GeneralHospital, Medical University of Vienna, Austria). Cells were grownin 75 cm2 culture flasks (Iwaki/Asahi Technoglass) as adherentmonolayer cultures in Minimal Essential Medium (MEM) supple-mented with 10% heat-inactivated fetal bovine serum, 1 mMsodium pyruvate, and 2 mM L-glutamine (all purchased from Sig-maeAldrich) without antibiotics. Cultures were maintained at37 �C in a humidified atmosphere containing 5% CO2 and 95% air.
4.6. Cytotoxicity tests in cancer cell lines
Cytotoxicity in the cell lines mentioned above was determinedby the colorimetric MTT assay (MTT ¼ 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, purchased fromFluka). Cells were harvested from culture flasks by trypsinizationand seeded in 100 mL aliquots in MEM supplemented with 10%heat-inactivated fetal bovine serum, 1 mM sodium pyruvate, 2 mML-glutamine, and 1% non-essential amino acids (100�) into 96-wellmicroculture plates (Iwaki/Asahi Technoglass) in the followingdensities, to ensure exponential growth of untreated controls
throughout the experiment: 1.5 � 103 (CH1), 3.5 � 103 (SK-OV-3),4.0 � 103 (A549), and 2.5 � 103 (SW480) viable cells per well. Cellswere allowed to settle and resume exponential growth in drug-freecomplete culture medium for 24 h, followed by the addition ofdilutions of the test compounds in 100 mL/well of the samemedium. After continuous exposure for 96 h, the medium wasreplaced by a 100 mL/well RPMI 1640 medium (supplemented with10% heat-inactivated fetal bovine serum and 4 mM L-glutamine)plus 20 mL/well solution of MTT in phosphate-buffered saline(5 mg/mL) (all purchased from SigmaeAldrich). After incubationfor 4 h, medium/MTT mixtures were removed, and the formazanproduct formed by viable cells was dissolved in DMSO (150 mL/well). Optical densities at 550 nmweremeasuredwith amicroplatereader (Tecan Spectra Classic), using a reference wavelength of690 nm to correct for unspecific absorption. The quantity of viablecells was expressed as percentage of untreated controls, and 50%inhibitory concentrations (IC50) were calculated fromconcentration-effect curves by interpolation. Evaluation is based onmeans from three independent experiments, each comprising sixreplicates per concentration level.
4.7. Apoptosis/necrosis assay
Cell death was analyzed by fluorescence-activated cell sorting(FACS) using FITC-conjugated annexin V (BioVision, USA) and pro-pidium iodide (PI; Fluka) staining (Table 6).
SW480 cells were seeded into 6-well plates (Iwaki/Asahi Tech-noglass, Gyouda, Japan) in amounts of 2 � 105 cells per well incompletemedium(as described above) and allowed to settle for 24 h.The cells were exposed to cisplatin and compound 7 for 48 h at 37 �C.After the incubation, cells were gently trypsinized, washed with PBS,and suspended with FITC-conjugated annexin V (0.25 mg/mL) inbinding buffer (10 mM HEPES/NaOH pH 7.4, 140 mM NaCl, 2.5 mMCaCl2) at room temperature for 15 min. PI (1 mg/mL) was addedshortly before the measurement. Stained cells were analyzed witha FACSCalibur instrument (BectonDickinson, Franklin Lakes,NJ, USA)using Cell-QuestPro software. At least three independent experi-ments were conducted, and 10,000 cells were counted per analysis.
Acknowledgments
H. V. is thankful for financial support of the University of Viennawithin the doctoral program “Initiativkolleg Functional Molecules”IKI041-N. The authors are indebted to the FFG e Austrian Research
Table 5Results from the calibration curves log P ¼ f (log k0) under different experimentalconditions.
%MeOH Equation R2
0 0.8842� �2.1484 0.9210 0.9126� �1.6351 0.9320 0.9586� �1.1251 0.9530 0.9736� �0.4783 0.9840 1.0174� þ0.081 0.9650 1.0063� þ0.7582 0.98
Table 6Viable, apoptotic and necrotic cell fractions (in %) of SW480 cells upon treatmentwith cisplatin or compound 7 for 48 h, analyzed by FACS using annexin V and PIstaining.
Concentration, mM Viable cells Earlyapoptosis
Lateapoptosis
Necrosis
Compound 7 (in %)0 98.3 � 0.6 0.7 � 0.3 0.8 � 0.5 0.2 � 0.15 97.2 � 1.2 1.5 � 0.8 1.0 � 0.4 0.4 � 0.310 87.9 � 9.8 3.3 � 1.9 5.1 � 5.0 3.8 � 3.715 77.7 � 8.3 3.9 � 2.9 10.6 � 5.9 7.9 � 2.625 62.4 � 4.9 5.8 � 4.9 14.7 � 6.2 17.1 � 7.850 47.5 � 2.6 4.8 � 0.8 26.3 � 8.0 21.5 � 7.0
Cisplatin (in %)0 98.1 � 0.7 0.7 � 0.6 0.5 � 0.4 0.7 � 0.55 96.1 � 2.1 1.0 � 0.2 1.1 � 0.7 1.9 � 2.210 93.5 � 2.0 2.5 � 0.8 1.5 � 1.2 2.5 � 3.115 95.9 � 1.0 2.9 � 0.3 1.0 � 0.6 0.2 � 0.125 94.9 � 3.5 3.3 � 3.4 1.3 � 0.6 0.6 � 0.550 93.8 � 3.5 3.7 � 2.6 1.6 � 0.8 0.9 � 0.7100 78.7 � 11.3 8.0 � 3.5 4.6 � 4.0 8.7 � 4.7
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Promotion Agency, the Austrian Council for Research and Tech-nology Development, the FWF (Austrian Science Fund) and COSTD39. We are thankful to Anna Bytzek for determination of log Pvalues via the shake-flask method. We are indebted to Prof. VerenaDirsch and Daniel Schachner (Institute of Pharmacognosy, Univer-sity of Vienna, Austria) for providing FACS equipment and technicalassistance.
Appendix. Supplementary material
Supplementary data related to this article can be found online atdoi:10.1016/j.ejmech.2011.09.006.
References
[1] M. Rosenberg, L. VanCamp, T. Krigas, Inhibition of cell division in Escherichiacoli by electrolysis products from a platinum electrode, Nature 205 (1965)698e699.
[2] N.J. Wheate, S. Walker, G.E. Craig, R. Oun, The status of platinum anticancerdrugs in the clinic and in clinical trials, Dalton Trans. 39 (2010) 8113e8127.
[3] M.D. Hall, H.R. Mellor, R. Callaghan, T.W. Hambley, Basis for design anddevelopment of platinum(IV) anticancer complexes, J. Med. Chem. 50 (15)(2007) 3403e3411.
[4] M. Galanski, Recent developments in the field of anticancer platinumcomplexes, Recent Pat. Anticancer Drug Discov. 1 (2006) 285e295.
[5] M. Galanski, M.J. Jakupec, B.K. Keppler, Update of the preclinical situation ofanticancer platinum complexes: novel design strategies and innovativeanalytical approaches, Curr. Med. Chem. 12 (18) (2005) 2075e2094.
[6] R.J. Schilder, F.P. LaCreta, R.P. Perez, S.W. Johnson, J.M. Brennan, A. Rogatko,S. Nash, C. McAleer, T.C. Hamilton, D. Roby, R.C. Young, R.F. Ozols, P.J. O’Dwyer,Phase I and pharmacokinetic study of ormaplatin (tetraplatin, NSC 363812)administered on a day 1 and day 8 schedule, Cancer Res. 54 (3) (1994) 709e717.
[7] G.B. Inc, Orplanta. Satraplatin Capsules, in Advisory Committee BriefingDocument. USA Food and Drug Administration, 2007.
[8] European Medicines Agency, Withdrawal Assessment Report for Orplanta(2008) 1e37.
[9] http://www.clinicaltrials.gov, U.S National Library ofMedicine, Bethesda, 1993.[10] O. Novakova, O. Vrana, V.I. Kiseleva, V. Brabec, DNA interactions of antitumor
platinum(IV) complexes, Eur. J. Biochem. 228 (3) (1995) 616e624.[11] M. Galanski, B.K. Keppler, Is reduction required for antitumor activity of
platinum(IV) compounds? Characterisation of a platinum(IV)-nucleotideadduct [enPt(OCOCH3)3(50-GMP)] by NMR spectroscopy and ESI-MS, Inorg.Chim. Acta 300e302 (2000) 783e789.
[12] L.T. Ellis, H.M. Er, T.W. Hambley, The influence of the axial ligands of a series ofplatinum(IV) anti-cancer complexes on their reduction to platinum(II) andreaction with DNA, Aust. J. Chem. 48 (4) (1995) 793e806.
[13] S. Choi, C. Filotto, M. Bisanzo, S. Delaney, D. Lagasee, J.L. Whitworth, A. Jusko,C. Li, N.A. Wood, J. Willingham, A. Schwenker, K. Spaulding, Reduction andanticancer activity of platinum(IV) complexes, Inorg. Chem. 37 (10) (1998)2500e2504.
[14] S.G. Chaney, S. Wyrick, G.K. Till, In vitro biotransformations of tetrachloro(d, l-trans)-1,2-diaminocyclohexaneplatinum(IV) (tetraplatin) in rat plasma,Cancer Res. 50 (1990) 4539e4545.
[15] L. Pendyala, J.W. Cowens, G.B. Chheda, S.P. Dutta, P.J. Creaven, Identification ofcis-dichloro-bis-isopropylamine platinum(II) as a major metabolite of ipro-platin in humans, Cancer Res. 48 (1988) 3533e3536.
[16] R.R. Barefoot, Speciation of platinum compounds: a review of recent appli-cations in studies of platinum anticancer drugs, J. Chromatogr. B. 751 (2001)205e211.
[17] M. Reithofer, M. Galanski, A. Roller, B.K. Keppler, An entry to novel platinumcomplexes: carboxylation of dihydroxoplatinum(IV) complexes with succinicanhydride and subsequent derivatization, Eur. J. Inorg. Chem. 13 (2006)2612e2617.
[18] M.R. Reithofer, S.M. Valiahdi, M.A. Jakupec, V.B. Arion, A. Egger, M. Galanski,B.K. Keppler, Novel di- and tetracarboxylatoplatinum(IV) complexes.Synthesis, characterization, cytotoxic activity, and DNA platination, J. Med.Chem. 50 (2007) 6692e6699.
[19] M.R. Reithofer, A. Schwarzinger, S.M. Valiahdi, M. Galanski, M.A. Jakupec,B.K. Keppler, Novel bis(carboxylato)dichlorido(ethane-1,2-diamine)plati-num(IV) complexes with exceptionally high cytotoxicity, J. Inorg. Biochem.102 (2008) 2072e2077.
[20] M.R. Reithofer, S.M. Valiahdi, M. Galanski, M.A. Jakupec, V.B. Arion,B.K. Keppler, Novel endothall containing platinum(IV) complexes e synthesis,characterization, and cytotoxic activity, Chem. Biodiv 5 (2008) 2160e2170.
[21] A. Bytzek,M. Reithofer,M. Galanski,M. Groessl, B.K. Keppler, C.G. Hartinger, Thefirst example of MEEKC-ICP-MS coupling and its application for the analysis ofanticancer platinum complexes, Electrophoresis 31 (2010) 1144e1150.
[22] M.R. Reithofer, A.K. Bytzek, S.M. Valiahdi, C.R. Kowol, M. Groessl,C.G. Hartinger, M.A. Jakupec, M. Galanski, B.K. Keppler, Tuning of lipophilicityand cytotoxic potency by structural variation of anticancer platinum(IV)complexes, J. Inorg. Biochem. 105 (2011) 46e51.
[23] W.H. Ang, S. Pilet, R. Scopelliti, F. Bussy, L. Juillerat-Jeanneret, P.J. Dyson,Synthesis and characterization of platinum(IV) anticancer drugs with func-tionalized aromatic carboxylate ligands: influence of the ligands on drugefficacies and uptake, J. Med. Chem. 48 (2005) 8060e8069.
[24] I.V. Tetko, I. Jaroszewicz, J.A. Platts, J. Kuduk-Jaworska, Calculation of lipo-philicity for Pt(II) complexes: experimental comparison of several methods,J. Inorg. Biochem. 102 (7) (2008) 1424e1437.
[25] S. Dhara, A rapid method for the synthesis of cis-[Pt(NH3)2Cl2], Indian J. Chem.8 (1970) 193e194.
[26] SAINT-Plus, Version 7.06a and APEX2. Bruker-Nonius AXS Inc., Madison, WI,2004.
[27] G.M. Sheldrick, A short history of SHELX, Acta Cryst. A64 (2008) 112e122.[28] G.K. Johnson, Report ORNL-5138. OAK Ridge National Laboratory, Oak Ridge,
TN, 1976.[29] OECD Guidelines for Testing of Chemicals, No. 107. OECD, Paris, 1995.
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2. Novel tetracarboxylatoplatinum(IV) complexes as carboplatin
prodrugs.
H.P. Varbanov, S.M. Valiahdi, C.R. Kowol, M.A. Jakupec, M. Galanski, B.K. Keppler,
Dalton Trans., 2012, 41, 14404-14415.
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DaltonTransactionsAn international journal of inorganic chemistry
www.rsc.org/dalton Volume 41 | Number 47 | 21 December 2012 | Pages 14213–1444
Galanski, Keppler Novel tetracarboxylatoplatinum(IV) complexes as carboplatin prodrugs
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Cite this: Dalton Trans., 2012, 41, 14404
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Novel tetracarboxylatoplatinum(IV) complexes as carboplatin prodrugs†
Hristo P. Varbanov, Seied M. Valiahdi, Christian R. Kowol, Michael A. Jakupec, Markus Galanski* andBernhard K. Keppler*
Received 26th June 2012, Accepted 26th July 2012DOI: 10.1039/c2dt31366a
It is widely accepted that platinum(IV) complexes act as prodrugs and have to be activated by reduction tothe respective platinum(II) analogs. Recently it could be shown that introduction of lipophilic carboxylatoligands in the axial position leads to diaminedichloridoplatinum(IV) compounds with exceptionally highcytotoxicity. With the aim of improving the antiproliferative properties of carboplatin, a series oftwenty-one novel Pt(IV) complexes, featuring the equatorial ligand sphere of carboplatin as well aslipophilic axial carboxylato ligands, was synthesized. In depth characterization is based on elementalanalysis, ESI-MS, ATR-IR, and multinuclear (1H, 13C, 15N, and 195Pt) NMR spectroscopy. Theircytotoxic activity in four cell lines (CH1, SK-OV-3, SW480, and A549), lipophilicity, electrochemistryand additionally the rate of reduction in the presence of ascorbic acid were investigated. In contrast toanalogous diaminedicarboxylatodichloridoplatinum(IV) compounds, the cytotoxicity of noveldiaminetetracarboxylato counterparts could not substantially be increased by simply enhancing theirlipophilic character. It seems that not only the reduction potential, but also the rate of reduction has atremendous influence on the cytotoxic properties. This has to be taken into account for the developmentof novel anticancer platinum(IV) agents.
Introduction
After Rosenberg’s serendipitous discovery1 opened up the way forthe introduction of metal complexes in antineoplastic chemo-therapy, several platinum(II) complexes (i.e. cisplatin, carboplatinand oxaliplatin) have become the mainstay of recent cancer treat-ment.2 Nowadays, research in that field has focused on platinum(IV)and nonplatinum metal complexes. It has been expected thatimprovement of the pharmacological and toxicological profile incomparison to platinum(II) based drugs could be achieved.3,4
In this context it is worth mentioning that cytotoxic effects ofplatinum complexes were first found for a Pt(IV) compound,namely (OC-6-22)-diamminetetrachloridoplatinum(IV), a closeanalogue of cisplatin.5 Platinum(IV) complexes are kineticallymore inert than their Pt(II) counterparts. Reduction underhypoxic conditions accompanied by the loss of two ligandsoccurs more readily than ligand exchange reactions. Their phys-icochemical characteristics broaden the possibilities for thedesign of novel metal-based drugs in various ways, e.g. modu-lation of pharmacokinetic properties, additional opportunities fortargeted therapy, oral administration, the prodrug concept, etc.6
So far, three Pt(IV) complexes were studied in clinical trials
(tetraplatin, iproplatin and satraplatin), but none of them gainedclinical approval.7 However, satraplatin currently entered newphase I and II clinical trials in combination regimens.8 Generally,Pt(IV) complexes act as prodrugs via activation by reduction invivo to the reactive Pt(II) species.9–12 This mode of actionexplains the importance of the rate and mechanism of reductionas well as the final reduction products. An optimal pharmaco-logical profile can be expected, provided that the prodrug is rela-tively stable in the blood stream and will preferably be reducedto the active platinum(II) species in the tumor cell or tissue. Ithas been shown that such redox behavior can be observed forPt(IV) complexes with axial carboxylato ligands.13 Consequently,platinum(IV) complexes were coupled to targeting moieties14–20
or to pharmacologically relevant molecules ameliorating thecytotoxic properties of the platinum(II) fragment.21–24
A series of cis-diam(m)inebis(carboxylato)dichloridoplatinum(IV)complexes with high cytotoxic potency has been publishedrecently.25–29 It was found that increasing the lipophilicity of theaxial ligands yields compounds with IC50 values in the nano-molar range.27,28 Whether the latter concept can be transferred tokinetically more inert platinum(II) analogs such as carboplatinhas not yet been investigated. Therefore, we intended to answerthe following question within this project: Is it possible to sig-nificantly increase the antiproliferative properties of carboplatinjust by synthesizing lipophilic prodrugs only differing in theaxial ligands?
In line with that task, twenty-one novel Pt(IV) complexes,designed as prodrugs for carboplatin, were synthesized and com-pletely characterized by elemental analysis, ESI-MS, ATR-IR,
†Electronic supplementary information (ESI) available. See DOI:10.1039/c2dt31366a
University of Vienna, Institute of Inorganic Chemistry, WaehringerStrasse 42, A-1090 Vienna, Austria. E-mail: [email protected],[email protected]; Fax: +43-1-4277-52680;Tel: +43-1-4277-52601
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and multinuclear NMR spectroscopy. Their cytotoxic activity infour human tumor cell lines, originating from ovarian carcinoma(CH1 and SK-OV-3), colon carcinoma (SW480) and non-smallcell lung cancer (A549), was evaluated by means of the MTTcolorimetric assay. In order to elucidate their biological behavior,lipophilicity as well as redox properties were determined.
Results and discussion
Synthesis
A general reaction scheme for the synthesis of novel complexesis shown in Fig. 1. The oxidation of carboplatin to the respectivedihydroxido Pt(IV) analogue (2) using H2O2 in aqueous solutionsat ambient temperature was performed successfully with yieldsover 90%. Complex 2 was subsequently converted to a series oftetracarboxylatoplatinum(IV) complexes using different cyclicanhydrides in DMF; complexes 3–6 were isolated with yieldsover 75%. The latter were used as the starting material for thesynthesis of a plethora of amide and ester derivatives viaactivation of the free carboxylic groups with CDI in dry DMF,followed by reaction with the corresponding amine or alcoholate/alcohol mixtures. Pure products were isolated with the help ofcolumn chromatography and/or recrystallization with moderateyields (around 20% for the esters and 30% for the amides).
Spectroscopic characterization
The novel complexes were fully characterized by elementalanalysis, one and two dimensional multinuclear NMR spec-troscopy (1H, 13C, 15N, 195Pt), ESI-MS and ATR-IR. The oxi-dation of carboplatin and the following carboxylations areaccompanied by significant shifts of the signals in the 195PtNMR spectra. A 195Pt resonance for the platinum(II) complex (1)was detected at −88 ppm; upon oxidation to the correspondingdihydroxidoplatinum(IV) analogue, the chemical shift was foundat 3268 ppm. Carboxylation of (2) resulted in a downfield shiftof around 290 ppm with signals for complexes 3–6 between3551 and 3567 ppm. Expectedly, further derivatization of 3–6had no influence on the position of the Pt signals; chemical
shifts of all novel target complexes ranged between 3551 and3568 ppm.
Normally, 195Pt NMR resonances are broad and unresolved;half height line widths of several hundred Hertz are common.Good signal to noise ratios are not easy to get in the case of195Pt NMR measurements. Consequently, large line broadeningfactors are used for processing of data in order to suppressunwanted noise (lb = 100 Hz, Fig. 2a). However, a fine structureof the signal is still visible which can be improved by decreasingthe line broadening factor to 10 Hz (Fig. 2b). When measuring a1H decoupled 195Pt spectrum, a nicely resolved quintet with a1J(14N, 195Pt) coupling constant of 205 Hz was obtained(Fig. 2c). The coupling of 14N and 195Pt with the protons of thecoordinated ammine ligands is visible in the 1H NMR spectrum
Fig. 2 195Pt NMR spectra of complex 6a processed with line broaden-ing factors of 100 Hz (a) and 10 Hz (b), respectively. 195Pt NMR spec-trum of 6a measured with proton decoupling (c).
Fig. 1 Synthesis of novel tetracarboxylatoplatinum(IV) complexes.
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as exemplarily shown for complex 6a (Fig. 3). The couplingconstants 1J(14N, 1H) = 53 Hz and 2J(195Pt, 1H) = 53 Hz are inthe expected range for platinum(IV) complexes.30
All signals in the 1H and 13C NMR spectra were found withexpected chemical shifts and their correct assignment was per-formed on the basis of two dimensional 1H1H COSY, 1H13CHSQC and 1H13C HMBC spectra.
For complexes 5, 5a and 5f a mixture of stereoisomers (RR :SS : RS = 1 : 1 : 2) was obtained due to the use of prochiral3-methylglutaric anhydride for esterification of 2. Nevertheless,this cannot be proven in the NMR spectra, as observed in pre-vious work from our group.27 Since the chiral information issituated on the axial ligands, which will be released during thereduction, complexes 5, 5a and 5f were investigated withoutfurther chiral separation. Structures of the complexes were alsoproved by ESI-MS spectra, measured both in the positive as wellas in the negative ion mode. The highest intensities wereobserved for peaks assigned to [M + Na+]+, [M + H+]+, [M − H+]−
and in some cases [M + Cl−]−. The m/z values as well as the iso-topic distribution were in accordance with the theoretical values.
Crystal structure of complex 4
X-ray diffraction on a sample of 4 afforded a data set that wasgenerally rather poor. However, the solution gave the gross struc-ture of the complex, as shown in Fig. S1 in the ESI.†
Lipophilicity
Lipophilicity is one of the most important physicochemical para-meters, characterizing the pharmacokinetic behavior and dedu-cing the ability of drugs and drug candidates to pass through cellmembranes. In particular this is true for platinum complexes(especially for Pt(IV)), which results in many efforts for estab-lishing a reliable method for determination and prediction of logP (octanol–water partition coefficient) values of platinum-basedcytostatics.31–36 Verifying lipophilicity with RP-HPLC, based onchromatographic retention indexes, has many advantages incomparison with the classic shake-flask method, like indepen-dence from the compound’s concentration, faster, robust andreproducible measurements, etc.37 Measuring the isocratic and
extrapolated retention factors log k30 and log kw can providerelevant information about the lipophilic properties of thecompounds even without converting them into log P values.38
In previous studies, we have shown that for cis-diam(m)inodi-chloridobis(carboxylato)platinum(IV) complexes cytotoxicity,cellular accumulation as well as DNA platination increase withincreasing log P within a series of analogues.28,39 Herein, wehave measured the lipophilicity of all new compounds with thehelp of RP-HPLC, using water/MeOH mobile phases. Theobtained retention factors (log kw and log k30) are summarized inTable 1.
All novel complexes were more lipophilic than their Pt(II)precursor (carboplatin) and its oxidized form (2); the latter log kvalues could not be determined under the conditions used,because their retention times were lower than that of thestandard, uracil.
Cytotoxicity in cancer cell lines
All novel compounds were tested in comparison to cisplatin andcarboplatin in four human tumor cell lines, originating fromovarian carcinoma (CH1, SK-OV-3), colon carcinoma (SW480)and non-small cell lung cancer (A549), by means of the colori-metric MTT microculture assay. All of these cell lines, exceptfor CH1 cells, are primarily cisplatin- and carboplatin-resistant(showing IC50 values about one order of magnitude higher thanthat in CH1 cells). IC50 values are summarized in Table 2, andcomplete concentration–effect curves in CH1 cells are depictedin the ESI (Fig. S2†).
All novel complexes showed cytotoxic activity in the cispla-tin-sensitive CH1 cell line, with IC50 values ranging between 7.7and 171 μM. However, cytotoxicity is lower in comparison tothe clinically established platinum drugs cisplatin and carbopla-tin (IC50 0.16 and 1.4 μM, respectively). Antiproliferativeactivity in cisplatin-resistant A549, SW480 and SK-OV-3 cancer
Fig. 3 NH3 signal of complex 6a in the 1H NMR spectrum.
Table 1 log k30 (log k, obtained with a mobile phase, containing 30%MeOH) and log kw (values, extrapolated to 0% MeOH) for the newcomplexes
Compound log kw log k30
3 1.07 −0.154 1.35 0.145 2.06 0.646 2.27 1.133a 1.61 0.403b 2.16 0.953c 2.94 1.533d 2.71 1.423e 4.20 2.383f 2.23 0.803g 1.68 0.043h 3.13 1.543i 3.67 2.033j 3.32 1.754a 1.86 0.694b 2.54 1.224f 2.10 0.905a 2.77 1.205f 2.92 1.306a 2.97 1.576f 3.06 1.69
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cells is generally negligible. In accordance with previousstudies,26,28 the complexes with free carboxylic groups (3, 4, 5and 6) exert the lowest antiproliferative activity, probably due totheir lower lipophilicity. The respective ester and amide deriva-tives are more cytotoxic, increasing with the lipophilicity of thealcoholate, resp. amide moiety. However, a substantial decreaseof IC50 values with increasing lipophilicity of the side chainscould not be observed. Contrary to our expectations, differencesin cytotoxicity of, e.g., methyl and propyl ester derivatives (3aand 3c) are relatively small (IC50 values differing by a factor of1.3). In the case of analogous complexes with two ethylamineand two chlorido ligands in the equatorial position, the propylester derivative is by more than one order of magnitude morecytotoxic than the methyl ester counterpart.28
A comparison of cytotoxicity of complex 3 and its ester andamide derivatives in the CH1 cell line is represented in Fig. 4.Overall, antiproliferative activity is dependent on lipophilicity.Esters (3d and 3e) featuring an isopropyl or butyl ester moietyand cyclopentyl and cyclohexyl amide derivatives (3h and 3i)show the lowest IC50 values. However, it is not possible to reachthe cytotoxic potency of carboplatin by increasing the lipophili-city of platinum(IV) analogues.
Variation of the spacer (Fig. 5) between the two carbonylatoms in the axial ligands of ester or amide derivatives revealedthat derivatization of 2 with a lipophilic anhydride tends to bemost favorable. However, the differences in cytotoxicity are nottremendous with increasing size of the spacer, which on theother hand decreases the solubility, and clear-cut structure–activity relationships with respect to the spacer cannot be formu-lated unequivocally.
A semi-logarithmic plot of log kw values (Table 1) versus IC50
values is shown in Fig. 6, demonstrating a rough correlation ofIC50 values with lipophilicity, but with poor overall determi-nation, indicating a considerable influence of additional factorson cytotoxicity. Recent studies on diam(m)inebis(carboxylato)dichloridoplatinum(IV) complexes have shown that with increas-ing lipophilicity, compounds with higher cytotoxicity comparedto their Pt(II) counterparts can be obtained.26–28 Nevertheless,there seem to be limits to this approach, as was demonstrated bythis series of novel diamminetetracarboxylatoplatinum(IV) com-plexes, all of which are less cytotoxic than their precursor carbo-platin. In order to find an explanation for this particularbehavior, additional studies with respect to the redox behavior ofthese complexes were performed.
Redox behavior
Electrochemistry. As already mentioned above, the redoxproperties of Pt(IV) complexes have a significant influence on
Fig. 4 Comparison of the IC50 values of complexes 3a–j and carbopla-tin in CH1 cells.
Fig. 5 Comparison of the IC50 values of complexes 3–6 and their pro-pylamide and methyl ester derivatives in CH1 cells.
Table 2 Cytotoxicity of the novel complexes in comparison tocisplatin and carboplatin in four human cancer cell lines
Compound
IC50a (μM)
CH1 A549 SW480 SK-OV-3
3 171 ± 1 >500 >500 >5003a 32 ± 10 >500 >500 >5003b 28 ± 4 >500 >500 >5003c 24 ± 5 >500 >500 >5003d 8.6 ± 1.7 >250 350 ± 39 —3e 11 ± 6 >500 181 ± 44 —3f 44 ± 8 >500 >500 >5003g 62 ± 26 >500 >500 —3h 15 ± 5 >500 >500 —3i 28 ± 2 >500 >500 —3j 31 ± 13 >500 >500 —4 114 ± 23 >500 >500 >5004a 33 ± 13 >500 >500 —4b 7.7 ± 1.4 >500 >250 —4f 89 ± 7 >500 >500 —5 128 ± 48 >500 >500 >5005a 23 ± 9 >500 >500 —5f 49 ± 13 >500 >500 —6 125 ± 35 >500 >500 >5006a 22 ± 8 >500 >500 —6f 33 ± 4 >500 >500 —1 (carboplatin) 1.4 ± 0.4 91 ± 10 85 ± 28 67 ± 11Cisplatin 0.16 ± 0.03 1.3 ± 0.4 3.5 ± 0.3 1.9 ± 0.3
a 50% Inhibitory concentrations in CH1, A549, SW480 and SK-OV-3cells in the MTT assay, 96 h exposure. Values are the means ± standarddeviations obtained from three independent experiments.
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their pharmacological profile. Fast reduction could result in deac-tivation of the respective platinum(II) species and high systemictoxicity, whereas very slow reduction could be responsible forthe lack of anticancer activity. When platinum(IV) complexesexhibit axial carboxylato ligands, the reduction potential is situ-ated in a medium range, which is believed to be a prerequisitefor the platinum(IV) prodrug strategy.
In order to elucidate the influence of modifications in the axialcarboxylato ligands on the redox potential and in last conse-quence on the cytotoxic properties, complexes 3, 3b, 3f, 4f and5f were investigated by cyclic voltammetry in DMF solution at ascan rate of 100 mV s−1 using 0.15 M [n-Bu4N][BF4] as the sup-porting electrolyte (Table 3, Fig. 7).
All complexes showed an irreversible reduction peak,common for Pt(IV) compounds (Fig. 7). The reduction potentialsof the investigated complexes were found to be at ca. −0.7 V vs.NHE featuring no significant deviation in dependency on thestructure of the axial ligand. These values are slightly morenegative compared to those of the dichloridoethylendiamine-platinum(IV) analogues which are reduced at around −0.6 V.39
Thus, the novel carboplatin prodrugs are harder to reduce andshould therefore exhibit a slightly lower antiproliferative potencycompared to the dichlorido compounds. However, as mentionedabove the IC50 values of novel complexes are much higher thanexpected.
Very recently, Gibson and Hambley et al. have shown that, inthe case of platinum(IV) complexes featuring a dicarboxylatoligand in the equatorial position, rates of reduction by ascorbatedo not necessarily go parallel to the electrochemical reductionpotentials.40
They concluded that the reduction by ascorbate is very slow inthe absence of coordinating hydroxido or chlorido ligands,which are capable of forming a bridge to the reducing agent andthereby facilitating the electron transfer. In line with this expla-nation, the tetracarboxylatoplatinum(IV) complexes in the presentstudy (lacking any Cl− or OH− ligands) should be reduced veryslowly.
Incubation with ascorbic acid
In order to judge the influence of the equatorial dicarboxylatoleaving ligand on the rate of reduction, complex 3f and itsethylenediaminedichlorido analogue (M1, the structure is shownin Fig. S3 in the ESI†) were incubated with a 25-fold excess ofascorbic acid. The fate of reduction was measured with the helpof 1H NMR spectroscopy in a D2O buffered solution at physio-logical pH.
Complex M1 was completely reduced after 24 h, with a halflife of ca. 5 h. However, complex 3f showed a very slowreduction rate; 50% of the complex were reduced after 3 weeks.Curves of the reduction (Fig. S4†) and comparative NMRspectra (Fig. S5 and S6) are given in the ESI.† The extremelyslow reduction of 3f might be the reason for its lower cyto-toxicity in vitro.
Conclusions
A series of novel carboplatin prodrugs was synthesized andcharacterized in detail by various analytical techniques. Cyto-toxicity was dependent on the lipophilicity of the axial ligandsbut could not be enhanced down to nanomolar concentrations asobserved recently for analogs featuring chlorido ligands insteadof 1,1-cyclobutanedicarboxylate. The reduction behavior in thepresence of ascorbic acid was investigated and revealed a dra-matic influence of the equatorial leaving group on the rate ofreduction and in last consequence on the antiproliferative
Fig. 6 Semi-logarithmic plot of lipophilicity (determined as log kwwith RP-HPLC) vs. cytotoxicity in CH1 cells (IC50) for the new com-plexes (succ – complex 3 and its derivatives, Glu – complex 4 and itsderivatives, MeGlu – complex 5 and its derivatives, and DiMeGlu –
complex 6 and its derivatives).
Table 3 Summary of the electrochemical data
Compound Ep/PtIV → PtII
3 −0.693b −0.703f −0.684f −0.745f −0.70
Potentials in V ± 0.02 vs. NHE measured at a scan rate of 100 mV s−1 in0.15 M [n-Bu4N][BF4]/DMF.
Fig. 7 Cyclic voltammogram of complex 3b in DMF containing0.15 M [n-Bu4N][BF4] at a scan rate of 100 mV s−1 using a glassycarbon working electrode.
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potential. Nevertheless, further investigations are needed in orderto get a detailed picture of the reduction process as well as of themode of action of platinum(IV) complexes.
Experimental
Materials and methods
All reagents and solvents were obtained from commercial suppli-ers, and were used without further purification. Methanol andethanol were dried, according to standard procedures. Water waspurified through reverse osmosis, followed by double distillation.For column chromatography, silica gel 60 (Fluka) was used.High purity water used for the HPLC experiments was obtainedfrom a Millipore Synergy 185 UV Ultrapure Water system (Mol-sheim, France).
1H, 13C, 15N, 195Pt and two-dimensional 1H1H COSY, 1H13Cand 1H15N HSQC, and 1H13C HMBC NMR spectra wererecorded with a Bruker Avance III 500 MHz NMR spectrometerat 500.32 (1H), 125.81 (13C), 107.55 (195Pt), and 50.70 MHz(15N) in DMF-d7 or D2O (in the case of carboplatin (1) and itsdihydroxido Pt(IV) analogue (2)) at ambient temperature, usingthe solvent residual peak for 1H and 13C as the internal reference.The splitting of proton resonances in the 1H NMR spectra isdefined as s = singlet, bs = broad singlet, d = doublet, t = triplet,and m = multiplet. 15N chemical shifts were referenced relativeto external NH4Cl, whereas
195Pt chemical shifts were referencedrelative to external K2[PtCl4] (see Fig. 8 for the NMR numberingscheme).
IR spectra were recorded on a Bruker Vertex 70 FT-IR spec-trometer (4000–400 cm−1) by using an ATR unit. Intensities ofreported IR bands are defined as br = broad, s = strong, m =medium, and w = weak. Electrospray ionization mass spec-trometry was carried out with a Bruker Esquire 3000 instrumentusing MeOH as the solvent. Elemental analyses were performedwith a Perkin-Elmer 2400 CHN-Elemental Analyser by theMicroanalytical Laboratory of the University of Vienna.
Synthesis
The general reaction scheme is given in Fig. 1. Synthesis of pre-cursors 1 and 2 is based on literature methods41,42 with somemodifications.
(SP-4-2)-Diammine(1,1-cyclobutanedicarboxylato)platinum(II)(1). Carboplatin was prepared, starting from K2PtCl4 via cis-Pt(NH3)2I2, its activation with AgNO3 (1.9 equiv.) and reactionof the formed diamminediaqua complex with the disodium salt(prepared in situ) of 1,1′-cyclobutanedicarboxylic acid (CBDA)(0.95 equiv.) in water. After reducing the solvent volume andcooling (to 4 °C), a white crystalline powder was collected,washed with small amounts of cold water and dried over P2O5
under vacuum. Yield: 56%. Mr = 371.25 g mol−1. Elementalanalysis, found: C 19.25, H 2.91, N 7.49. Calcd forC6H12N2O4Pt: C 19.41, H 3.26, N 7.55. 1H NMR: δ = 2.79(t, 3JH,H = 7.9 Hz, 4H, H-2), 1.81 (m, 2H, H-1) ppm. 195Pt NMR:δ = −88 ppm. IR (ATR): 3258 s, 3190 br (νN–H); 2957 w; 1634 s,1601 s (νCvO); 1464 w; 1373 w, 1345 s, 1286 m, 1204 w cm−1.
(OC-6-33)-Diammine(cyclobutane-1,1-dicarboxylato)dihydro-xidoplatinum(IV) (2). Carboplatin (1) (1.4862 g, 4.0038 mmol)was oxidized with 20 ml of 30% H2O2 in 20 ml of water at roomtemperature. The white product formed was filtered off, washedwith cold water and dried over P2O5 under vacuum. The volumeof the filtrate was reduced, cooled in the fridge for 24 h and thenewly formed precipitate was also collected and dried. Yield:1.5169 g, 93%. Mr = 405.26 g mol−1. 1H NMR: δ = 2.59 (t,3JH,H = 8.1 Hz, 4H, H-2), 1.96 (m, 2H, H-1) ppm. 13C NMR:δ = 180.7 (C-4), 55.8 (C-3), 32.1 (C-2), 15.7 (C-1) ppm. 195Pt NMR:δ = 3268 ppm. IR (ATR): 3448 br (νPtO–H); 3217 br, 3093 br (νN–H);2963 br, 2727 w; 1615 s, 1570 s (νCvO); 1351 s cm−1.
General procedure for synthesis of compounds 3–6. 4 equiv.of the corresponding anhydride and (OC-6-33)-diammine(cyclo-butane-1,1-dicarboxylato)dihydroxidoplatinum(IV) (2) were sus-pended in dry DMF and the reaction mixture was stirred at 50 °Cuntil the solid material dissolved to form a colorless to paleyellowish solution. DMF was then removed under reducedpressure (a white foam is formed). The residue was suspended inacetone, filtered off and dried under vacuum to obtain a whitesolid.
(OC-6-33)-Diamminebis(3-carboxypropanoato)(cyclobutane-1,1-dicarboxylato)platinum(IV) (3). Succinic anhydride (215 mg,2.1500 mmol), 218 mg (0.5383 mmol) of 2 in DMF (6 mL),2 h. Yield: 278 mg, 85%. Mr = 605.4 g mol−1. Elemental analy-sis, found: C 27.95, H 3.51, N 4.62. Calcd for C14H22N2O12Pt:C 27.77, H 3.66, N 4.63. ESI-MS: m/z 627.1 [M + Na+]+, 605.1[M + H+]+, 603.9 [M − H+]−. 1H NMR: δ = 12.35 (bs, 2H,COOH), 6.73 (m, 6H, NH3), 2.64 (t, 3JH,H = 8.0 Hz, 4H, H-2),2.53 (t, 3JH,H = 6.9 Hz, 4H, H-6), 2.47 (t, 3JH,H = 6.6 Hz, 4H,H-7), 1.90 (m, 2H, H-1) ppm. 13C NMR: δ = 179.5 (C-5), 176.7(C-4), 173.8 (C-8), 56.4 (C-3), 31.9 (C-2), 30.3 (3JC,Pt = 37 Hz,C-6), 29.8 (C-7), 16.0 (C-1) ppm. 15N NMR: δ = −53.8 ppm.195Pt NMR: δ = 3565 ppm. IR (ATR): 3237 br, 3161 br (νN–H);2949 w; 1767 w, 1719 m 1632 s, 1594 m, 1546 w (νCvO);1407 w, 1349 s, 1328 s, 1286 w, 1178 m cm−1.
Fig. 8 NMR numbering scheme for novel tetracarboxylatoplatinum(IV)complexes.
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(OC-6-33)-Diamminebis(4-carboxybutanoato)(cyclobutane-1,1-dicarboxylato)platinum(IV) (4). Glutaric anhydride (350 mg,3.0675 mmol), 306 mg (0.7551 mmol) of 2 in DMF (8 mL),3 h. The product was recrystallized from methanol, washed withacetone and dried under vacuum. Yield: 384 mg, 83%. Mr =633.46 g mol−1. Elemental analysis, found: C 30.24, H 4.00,N 4.29. Calcd for C16H26N2O12Pt: C 30.34, H 4.14, N 4.42.ESI-MS: m/z 654.9 [M + Na+]+, 633.9 [M + H+]+, 630.8[M − H+]−. 1H NMR: δ = 12.25 (bs, 2H, COOH), 6.78 (m, 6H,NH3), 2.62 (t, 3JH,H = 8.0 Hz, 4H, H-2), 2.30 (t, 3JH,H = 7.6 Hz,4H, H-7), 2.28 (t, 3JH,H = 7.6 Hz, 4H, H-6), 1.90 (m, 2H, H-1),1.73 (m, 4H, H-7′) ppm. 13C NMR: δ = 180.1 (C-5), 176.8(C-4), 174.4 (C-8), 56.3 (C-3), 34.5 (C-6), 33.0 (C-7), 31.9(C-2), 21.0 (C-7′), 16.0 (C-1) ppm. 15N NMR: δ = −54.5 ppm.195Pt NMR: δ = 3551 ppm. IR (ATR): 3196 br, 3096 br (νN–H);2953 m; 1698 m, 1637 s (νCvO); 1411 w, 1332 s, 1234 m, 1087w cm−1.
(OC-6-33)-Diamminebis(4-carboxy-3-methylbutanoato)(cyclo-butane-1,1-dicarboxylato)platinum(IV) (5). 3-Methylglutaricanhydride (528 mg, 4.1208 mmol), 411 mg (1.0141 mmol) of 2in DMF (10 mL), 2 h. Yield: 520 mg, 78%. Mr = 661.52 gmol−1. Elemental analysis, found: C 32.62, H 4.48, N 4.22.Calcd for C18H30N2O12Pt: C 32.68, H 4.57, N 4.23. ESI-MS:m/z 699.1 [M + K+]+, 683.3 [M + Na+]+, 662.2 [M + H+]+,660.1 [M − H+]−. 1H NMR: δ = 12.23 (bs, 2H, COOH), 6.80(m, 6H, NH3), 2.63 (t, 3JH,H = 7.9 Hz, 4H, H-2), 2.37 (m, 2H,H-7), 2.32 (m, 2H, H-6), 2.29 (m, 2H, H-7′), 2.12 (m, 2H, H-6),2.08 (m, 2H, H-7), 1.90 (m, 2H, H-1), 0.93 (d, 3JH,H = 6.5 Hz,6H, H-7′′) ppm. 13C NMR: δ = 179.7 (C-5), 176.8 (C-4), 173.8(C-8), 56.3 (C-3), 42.2 (C-6), 40.6 (C-7), 31.9 (C-2), 27.8(C-7′), 19.4 (C-7′′), 16.0 (C-1) ppm. 15N NMR: δ = −55.3 ppm.195Pt NMR: δ = 3559 ppm. IR (ATR): 3209 br, 3093 br (νN–H);2963 w; 1704 m, 1637 s (νCvO); 1458 w, 1342 s, 1221 m, 1169w, 1080 w cm−1.
(OC-6-33)-Diamminebis(4-carboxy-3,3-dimethylbutanoato)-(cyclobutane-1,1-dicarboxylato)platinum(IV) (6). 3,3-Dimethyl-glutaric anhydride (212 mg, 1.4194 mmol), 151 mg(0.3726 mmol) of 2 in DMF (6 mL), 4 h. Yield: 210 mg, 82%.Mr = 689.56 g mol−1. Elemental analysis, found: C 34.88,H 4.88, N 4.08. Calcd for C20H34N2O12Pt: C 34.84, H 4.97,N 4.06. ESI-MS: m/z 712.2 [M + Na+]+, 688.7 [M − H+]−.1H NMR: δ = 12.14 (bs, 2H, COOH), 6.80 (m, 6H, NH3), 2.63(t, 3JH,H = 7.9 Hz, 4H, H-2), 2.34 (s, 4H, H-7), 2.32 (s, 4H,H-6), 1.90 (m, 2H, H-1), 1.06 (s, 12H, H-7′′) ppm. 13C NMR:δ = 179.4 (C-5), 176.8 (C-4), 173.2 (C-8), 56.3 (C-3), 46.9(C-6), 45.1 (C-7), 32.3 (C-7′), 31.9 (C-2), 27.0 (C-7′′), 16.0(C-1) ppm. 15N NMR: δ = −55.1 ppm. 195Pt NMR: δ =3567 ppm. IR (ATR): 3255 br, 3128 br (νN–H); 2964 m, 2879 w;1712 m, 1697 m, 1638 s, 1611 s (νCvO); 1365 s, 1345 s, 1229 s,1159 m, 1108 w cm−1.
General procedure for synthesis of ester derivatives 3a–e, 4a–b,5a and 6a
2 equiv. of CDI (1,1′-carbonyldiimidazole) in dry DMF wereadded to a DMF solution of 3, 4, 5 or 6, respectively, and themixture was heated at 60°C for 10 min. After cooling to room
temperature, CO2 formed during the activation was removed byflushing the solution with argon for 10 min. Then sodium alco-holate in absolute alcohol (prepared in situ by dissolving a cata-lytic amount of Na in absolute alcohol) was added and thesolution was stirred for 24 h (up to 72 h in some cases). The sol-vents were removed under reduced pressure and the residuespurified by recrystallization or column chromatography.
(OC-6-33)-Diammine(cyclobutane-1,1-dicarboxylato)bis-((4-methoxy)-4-oxobutanoato)platinum(IV) (3a). CDI (136 mg,0.9004 mmol) in DMF (5 mL), 3 (248.5 mg, 0.4105 mmol) inDMF (6 mL), MeONa/MeOH (10 mL). The crude product waspurified by suspending in EtOAc, filtration of the yellow powderand recrystallization of the latter from water. The obtained whitepowder was filtered off, washed with a small amount of coldwater and cold diethyl ether and dried in a vacuum desiccatorover P2O5. Yield: 60 mg, 23%. Mr = 633.46 g mol−1. Elementalanalysis, found: C 30.18, H 3.90, N 4.43. Calcd forC16H26N2O12Pt: C 30.34, H 4.14, N 4.42. ESI-MS: m/z 655.0[M + Na+]+, 633.9 [M + H+]+, 632.5 [M − H+]−, 669.0 [M +Cl−]−. 1H NMR: δ = 6.74 (m, 6H, NH3), 3.63 (s, 6H, H-9), 2.63(t, 3JH,H = 8.0 Hz, 4H, H-2), 2.54 (m, 4H, H-6), 2.48 (m, 4H,H-7), 1.90 (m, 2H, H-1) ppm. 13C NMR: δ = 179.2 (C-5), 176.7(C-4), 172.9 (C-8), 56.3 (C-3), 51.1 (C-9), 31.8 (C-2), 30.2(C-6), 29.5 (C-7), 15.9 (C-1) ppm. 15N NMR: δ = −54.0 ppm.195Pt NMR: δ = 3566 ppm. IR (ATR): 3256 br, 3204 br (νN–H),3119 br; 2953 w; 1732 m, 1639 s (νCvO); 1428 w 1315s,1262 w, 1191 w, 1164 m cm−1.
(OC-6-33)-Diammine(cyclobutane-1,1-dicarboxylato)bis-((4-ethoxy)-4-oxobutanoato)platinum(IV) (3b). CDI (223 mg,1.3753 mmol) in DMF (6 mL), 3 (416 mg, 0.6871 mmol) inDMF (8 mL), EtONa/EtOH (10 mL). The crude product waspurified by column chromatography (EtOAc/MeOH, 5 : 1), thenisolated through suspending in EtOAc, filtration and washingwith Et2O. A white powder was obtained and dried undervacuum. Yield: 130 mg, 31%. Mr = 661.51 g mol−1. Elementalanalysis, found: C 32.49, H 4.47, N 4.31. Calcd forC18H30N2O12Pt: C 32.68, H 4.57, N 4.23. ESI-MS: m/z 699.0[M + K+]+, 683.0 [M + Na+]+, 662.0 [M + H+]+, 659.7 [M −H+]−, 696.7 [M + Cl−]−. 1H NMR: δ = 6.73 (m, 6H, NH3), 4.08(m, 4H, H-9), 2.63 (t, 3JH,H = 8.0 Hz, 4H, H-2), 2.53 (m, 4H,H-6), 2.47 (m, 4H, H-7), 1.90 (m, 2H, H-1), 1.20 (t, 3JH,H =7.1 Hz, 6H, H-10) ppm. 13C NMR: δ = 179.3 (C-5), 176.7(C-4), 172.4 (C-8), 56.3 (C-3), 60.0 (C-9), 31.8 (C-2), 30.2(C-6), 29.8 (C-7), 15.9 (C-1), 13.8 (C-10) ppm. 15N NMR: δ =−54.8 ppm. 195Pt NMR: δ = 3564 ppm. IR (ATR): 3251 br,3208 br (νN–H); 2980 w; 1727 m, 1640 s (νCvO); 1424 w,1317 s, 1093 m cm−1.
(OC-6-33)-Diammine(cyclobutane-1,1-dicarboxylato)bis-((4-propyloxy)-4-oxobutanoato)platinum(IV) (3c). CDI (0.2143 g,1.3214 mmol) in DMF (9 mL), 3 (0.4000 g, 0.6607 mmol) inDMF (10 mL), n-PrONa/n-PrOH (15 mL). The crude productwas purified by column chromatography (EtOAc/IPA, 3 : 1), thenisolated from an Et2O suspension, followed by MeOH/EtOAcrecrystallization. The white powder obtained was dried undervacuum. Yield: 82 mg, 18%. Mr = 689.57 g mol−1. Elementalanalysis, found: C 34.70, H 4.74, N 4.04. Calcd forC20H34N2O12Pt: C 34.84, H 4.97, N 4.06. ESI-MS: m/z 734.0
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[M + 2Na+ − H+]+, 711.1 [M + Na+]+, 689.9 [M + H+]+, 687.9[M − H+]−. 1H NMR: δ = 6.74 (m, 6H, NH3), 3.99 (t, 3JH,H =6.7 Hz, 4H, H-9), 2.63 (t, 3JH,H = 8.0 Hz, 4H, H-2), 2.54 (m,4H, H-6), 2.48 (m, 4H, H-7), 1.90 (m, 2H, H-1), 1.61 (m, 4H,H-10), 0.91 (t, 3JH,H = 7.4 Hz, 6H, H-11) ppm. 13C NMR: δ =179.3 (C-5), 176.7 (C-4), 172.5 (C-8), 65.6 (C-9), 56.3 (C-3),31.8 (C-2), 30.2 (C-6), 29.8 (C-7), 21.8 (C-10), 15.9 (C-1), 9.9(C-11) ppm. 15N NMR: δ = −54.1 ppm. 195Pt NMR: δ =3565 ppm. IR (ATR): 3245 br, 3210 br (νN–H); 2969 w; 1730 m,1641 s (νCvO); 1318 s, 1259 w, 1090 m cm−1.
(OC-6-33)-Diammine(cyclobutane-1,1-dicarboxylato)bis((4-(2-propyloxy))-4-oxobutanoato)platinum(IV) (3d). CDI (230 mg,1.4184 mmol) in DMF (10 mL), 3 (411 mg, 0.6789 mmol) inDMF (9 mL), i-PrONa/i-PrOH (10 mL). The crude product waspurified by column chromatography (EtOAc/IPA, 2 : 1), then iso-lated from an EtOAc suspension, washed with cold Et2O anddried under vacuum to obtain a white powder. Yield: 36 mg, 8%.Mr = 689.57 g mol−1. Elemental analysis, found: C 33.58,H 4.86, N 4.08. Calcd for C20H34N2O12Pt·H2O: C 33.95,H 5.13, N 3.96. ESI-MS: m/z 711.0 [M + Na+]+, 687.8 [M −H+]−. 1H NMR: δ = 6.74 (m, 6H, NH3), 4.91 (m, 2H, H-9), 2.63(t, 3JH,H = 8.0 Hz, 4H, H-2), 2.52 (m, 4H, H-6), 2.44 (m, 4H,H-7), 1.90 (m, 2H, H-1), 1.20 (d, 3JH,H = 6.3 Hz, 12H, H-10)ppm. 13C NMR: δ = 179.3 (C-5), 176.7 (C-4), 171.9 (C-8), 67.4(C-9), 56.3 (C-3), 31.8 (C-2), 30.3 (C-6), 30.1 (C-7), 21.3 (C-10),15.9 (C-1) ppm. 15N NMR: δ = −54.1 ppm. 195Pt NMR: δ =3565 ppm. IR (ATR): 3230 br (νN–H); 2980 w; 1728 m, 1654 s,1630 s (νCvO); 1339 s, 1255 w, 1205 w, 1172 m, 1105 s cm−1.
(OC-6-33)-Diammine(cyclobutane-1,1-dicarboxylato)bis-((4-butyloxy)-4-oxobutanoato)platinum(IV) (3e). CDI (224 mg,1.3814 mmol) in DMF (9 mL), 3 (400 mg, 0.6607 mmol) inDMF (10 mL), n-ButONa/n-ButOH (10 mL). The crude productwas purified by column chromatography (EtOAc/MeOH, 9 : 1),then recrystallized from MeOH, and dried under vacuum toobtain a white powder. Yield: 110 mg, 11%. Mr = 717.62 gmol−1. Elemental analysis, found: C 36.60, H 5.14, N 3.93.Calcd for C22H38N2O12Pt: C 36.82, H 5.34, N 3.90. ESI-MS:m/z 739.2 [M + Na+]+, 716.1 [M − H+]−. 1H NMR: δ = 6.73 (m,6H, NH3), 4.04 (t, 3JH,H = 6.7 Hz, 4H, H-9), 2.63 (t, 3JH,H =8.0 Hz, 4H, H-2), 2.54 (m, 4H, H-6), 2.48 (m, 4H, H-7), 1.90(m, 2H, H-1), 1.58 (m, 4H, H-10), 1.36 (m, 4H, H-11), 0.91 (t,3JH,H = 7.4 Hz, 6H, H-12) ppm. 13C NMR: δ = 179.3 (C-5),176.7 (C-4), 172.5 (C-8), 63.9 (C-9), 56.3 (C-3), 31.8 (C-2),30.6 (C-10), 30.2 (C-6), 29.8 (C-7), 18.9 (C-11), 15.9 (C-1),13.3 (C-12) ppm. 15N NMR: δ = −54.1 ppm. 195Pt NMR: δ =3565 ppm. IR (ATR): 3305 br, 3236 br (νN–H); 2959 w; 1728 m,1616 s, 1571 s (νCvO); 1424 m, 1399 s, 1169 m cm−1.
(OC-6-33)-Diammine(cyclobutane-1,1-dicarboxylato)bis-((5-methoxy)-5-oxopentanoato)platinum(IV) (4a). CDI (196 mg,1.209 mmol) in DMF (8 mL), 4 (360 mg, 0.568 mmol) in DMF(8 mL), MeONa/MeOH (10 mL). The crude product waspurified by column chromatography (EtOAc/MeOH, 3 : 1) andisolated via suspending in Et2O, filtration, washing with Et2O,EtOAc and diisopropylester and drying under vacuum to obtaina white powder. Yield: 94 mg, 16%. Mr = 661.51 g mol−1.Elemental analysis, found: C 32.33, H 4.32, N 4.26. Calcd forC18H30N2O12Pt: C 32.68, H 4.57, N 4.23. ESI-MS: m/z 683.1
[M + Na+]+, 661.0 [M + H+]+, 659.9 [M − H+]−. 1H NMR: δ =6.76 (m, 6H, NH3), 3.63 (s, 6H, H-9), 2.62 (t, 3JH,H = 8.0 Hz,4H, H-2), 2.34 (t, 3JH,H = 7.5 Hz, 4H, H-7), 2.27 (t, 3JH,H =7.4 Hz, 4H, H-6), 1.90 (m, 2H, H-1), 1.74 (m, 4H, H-7′) ppm.13C NMR: δ = 180.0 (C-5), 176.8 (C-4), 173.4 (C-8), 56.3 (C-3),50.9 (C-9), 34.4 (C-6), 32.7 (C-7), 31.9 (C-2), 20.9 (C-7′), 16.0(C-1) ppm. 15N NMR: δ = −54.4 ppm. 195Pt NMR: δ =3555 ppm. IR (ATR): 3240 br, 3210 br (νN–H); 2952 w; 1733 m,1639 s (νCvO); 1436 w, 1355 s, 1241 m, 1202 m, 1148 m cm−1.
(OC-6-33)-Diammine(cyclobutane-1,1-dicarboxylato)bis-((5-ethoxy)-5-oxopentanoato)platinum(IV) (4b). CDI (112.1 mg,0.6913 mmol) in DMF (7 mL), 4 (219 mg, 0.3457 mmol) inDMF (5 mL), EtONa/EtOH (8 mL). The crude product waspurified by column chromatography (EtOAc/IPA, 2 : 1) and iso-lated via suspending in EtOAc, followed by recrystallizationfrom MeOH/EtOAc and final washing with cold Et2O. Thewhite powder obtained was dried under vacuum. Yield: 42 mg,18%. Mr = 689.57 g mol−1. Elemental analysis, found: C 33.98,H 4.65, N 3.92. Calcd for C20H34N2O12Pt·0.5H2O: C 34.38,H 5.05, N 4.01. ESI-MS: m/z 712.0 [M + Na+]+, 690.0 [M +H+]+, 686.8 [M − H+]−. 1H NMR: δ = 6.77 (m, 6H, NH3), 4.09(m, 4H, H-9), 2.63 (t, 3JH,H = 7.9 Hz, 4H, H-2), 2.33 (t, 3JH,H =7.5 Hz, 4H, H-7), 2.28 (t, 3JH,H = 7.4 Hz, 4H, H-6), 1.90 (m,2H, H-1), 1.74 (m, 4H, H-7′), 1.21 (t, 3JH,H = 7.1 Hz, 6H, H-10)ppm. 13C NMR: δ = 180.1 (C-5), 176.9 (C-4), 173.0 (C-8), 59.9(C-9), 56.3 (C-3), 34.4 (C-6), 33.0 (C-7), 31.8 (C-2), 21.0(C-7′), 16.0 (C-1), 13.9 (C-10) ppm. 15N NMR: δ = −55.2 ppm.195Pt NMR: δ = 3554 ppm. IR (ATR): 3198 br (νN–H); 2980 w;1733 w, 1718 m, 1622 s (νCvO); 1416 w, 1358 s, 1327 s,1207 m, 1106 m cm−1.
(OC-6-33)-Diammine(cyclobutane-1,1-dicarboxylato)bis-((5-methoxy)-5-oxo-(3-methyl)pentanoato)platinum(IV) (5a). CDI(240 mg, 1.480 mmol) in DMF (10 mL), 5 (481 mg,0.727 mmol) in DMF (10 mL), MeONa/MeOH (10 mL). Thecrude product was purified by column chromatography (EtOAc/MeOH, 5 : 1) and isolated via suspending in EtOAc, filtration,washing with Et2O and EtOAc and drying under vacuum toobtain a white powder. Yield: 192 mg, 38%. Mr = 689.57 gmol−1. Elemental analysis, found: C 34.74, H 4.76, N 4.04.Calcd for C20H34N2O12Pt: C 34.84, H 4.97, N 4.06. ESI-MS:m/z 726.9 [M + K+]+, 711.1 [M + Na+]+, 689.9 [M + H+]+,687.8 [M − H+]−, 723.9 [M + Cl−]−. 1H NMR: δ = 6.78 (m, 6H,NH3), 3.63 (s, 6H, H-9), 2.63 (t, 3JH,H = 8.0 Hz, 4H, H-2), 2.44(m, 2H, H-7), 2.29 (m, 2H, H-6), 2.27 (m, 2H, H-7′), 2.15 (m,2H, H-6), 2.13 (m, 2H, H-7), 1.90 (m, 2H, H-1), 0.91 (d, 3JH,H =6.5 Hz, 6H, H-7′′) ppm. 13C NMR: δ = 179.5 (2JC,Pt = 28 Hz, C-5),176.8 (C-4), 172.8 (C-8), 56.3 (C-3), 50.9 (C-9), 42.0 (3JC,Pt = 36Hz, C-6), 40.2 (C-7), 31.9 (C-2), 27.8 (C-7′), 19.3 (C-7′′), 16.0(C-1) ppm. 15N NMR: δ = −54.6 ppm. 195Pt NMR: δ = 3560 ppm.IR (ATR): 3239 br, 3094 br (νN–H); 2876 w; 1732 m, 1632 s(νCvO); 1437 w, 1331 s, 1211 m, 1155 m, 1079 m cm−1.
(OC-6-33)-Diammine(cyclobutane-1,1-dicarboxylato)bis-((5-methoxy)-5-oxo-(3,3-dimethyl)pentanoato)platinum(IV) (6a).CDI (180 mg, 1.110 mmol) in DMF (6 mL), 6 (375 mg,0.544 mmol) in DMF (10 mL), MeONa/MeOH (8 mL). Thecrude product was purified by column chromatography (EtOAc/MeOH, 6 : 1) and isolated via suspending in EtOAc, filtration,
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washing with Et2O and drying under vacuum to obtain a whitepowder. Yield: 125 mg (32%). Mr = 717.62 g mol−1. Elementalanalysis, found: C 36.51, H 5.35, N 3.87. Calcd forC22H38N2O12Pt: C 36.82, H 5.34, N 3.90. ESI-MS: m/z 761.8[M + 2Na+ − H+]+, 739.9 [M + Na+]+, 715.9 [M − H+]−.1H NMR: δ = 6.79 (m, 6H, NH3), 3.61 (s, 6H, H-9), 2.63 (t,
3JH,H =7.9 Hz, 4H, H-2), 2.39 (s, 4H, H-7), 2.29 (s, 4H, H-6), 1.91 (m,2H, H-1), 1.03 (s, 12H, H-7′′) ppm. 13C NMR: δ = 179.2 (C-5),176.8 (C-4), 172.2 (C-8), 56.3 (C-3), 50.6 (C-9), 46.8 (3JC,Pt =36 Hz, C-6), 44.6 (C-7), 32.5 (C-7′), 31.9 (C-2), 26.9 (C-7′′),16.0 (C-1) ppm. 15N NMR: δ = −55.8 ppm. 195Pt NMR: δ =3567 ppm. IR (ATR): 3230 br, 3097 br (νN–H); 2956 m, 2876 w;1735 m, 1618 s, 1570 w (νCvO); 1436 w, 1332 s, 1227 m,1149 m, 1112 m cm−1.
General procedure for synthesis of amide derivatives 3f–j, 4f,5f, 6f
2 equiv. of CDI (1,1′-carbonyldiimidazole) in dry DMF wereadded to a DMF solution of 3, 4, 5 or 6, respectively, and themixture was heated at 60 °C for 10 min. After cooling to roomtemperature, CO2 formed during the activation was removed byflushing the solution with argon for 10 min. Then, 2.3 equiv. ofthe corresponding amine, dissolved in dry DMF, were added andthe solution was stirred for 24 h (up to 48 h in some cases).Finally, DMF was removed under reduced pressure and thecrude products were purified by column chromatography.
(OC-6-33)-Diammine(cyclobutane-1,1-dicarboxylato)bis-((4-propylamino)-4-oxobutanoato)platinum(IV) (3f). CDI (218 mg,1.3444 mmol) in DMF (8 mL), 3 (407 mg, 0.6723 mmol) inDMF (8 mL), propylamine (112 μl, 1.3567 mmol) in DMF(2 mL). The crude product was purified by column chromato-graphy (EtOAc/MeOH, 2 : 1) and isolated from an EtOAc sus-pension, followed by recrystallization from MeOH and washingwith a cold mixture of EtOAc and Et2O. An almost white to paleyellow powder was obtained and dried under vacuum. Yield:124 mg, 24%. Mr = 687.20 g mol−1. Elemental analysis, found:C 34.43, H 5.21, N 8.15. Calcd for C20H36N4O10Pt·0.5H2O:C 34.48, H 5.35, N 8.04. ESI-MS: m/z 709.9 [M + Na+]+, 685.9[M − H+]−. 1H NMR: δ = 7.78 (bs, 2H, CONH), 6.73 (m, 6H,NH3), 3.09 (m, 4H, H-9), 2.62 (t, 3JH,H = 8.0 Hz, 4H, H-2), 2.48(t, 3JH,H = 7.1 Hz, 4H, H-6), 2.35 (t, 3JH,H = 7.6 Hz, 4H, H-7),1.89 (m, 2H, H-1), 1.46 (m, 4H, H-10), 0.87 (t, 3JH,H = 7.4 Hz,6H, H-11) ppm. 13C NMR: δ = 180.1 (C-5), 176.8 (C-4), 171.5(C-8), 56.3 (C-3), 40.8 (C-9), 31.8 (C-2), 31.6 (C-7), 31.2 (C-6),22.7 (C-10), 16.0 (C-1), 11.0 (C-11) ppm. 15N NMR: δ = −54.3(NH3), 94.1 (NH-amide) ppm. 195Pt NMR: δ = 3562 ppm. IR(ATR): 3236 br, 3087 br (νN–H); 2963 w; 1625 s, 1551 m(νCvO); 1333 s, 1249 m, 1207 m, 1158 w cm−1.
(OC-6-33)-Diammine(cyclobutane-1,1-dicarboxylato)bis((4-(2-methoxy)ethylamino)-4-oxobutanoato)platinum(IV) (3g). CDI(159 mg, 0.9806 mmol) in DMF (5 mL), 3 (283 mg,0.4675 mmol) in DMF (5 mL), 2-methoxyethylamine (101 μl,1.1726 mmol) in DMF (4 mL). The crude product was purifiedby column chromatography (EtOAc/MeOH, 1 : 1). A yellowsticky substance was isolated and purified again by columnchromatography, using EtOAc/MeOH = 3 : 1 as mobile phase.
The final product was obtained as a white powder through sus-pension in EtOAc, filtration, washing with Et2O and dryingunder vacuum. Yield: 72 mg, 21%. Mr = 719.60 g mol−1.Elemental analysis, found: C 32.86, H 4.90, N 7.47. Calcd forC20H36N4O12Pt·0.5H2O: C 32.97, H 5.12, N 7.69. ESI-MS: m/z741.9 [M + Na+]+, 720.0 [M + H+]+, 718.1 [M − H+]−. 1HNMR: δ = 7.86 (bs, 2H, CONH), 6.72 (m, 6H, NH3), 3.39 (t,3JH,H = 5.9 Hz, 4H, H-10), 3.31 (t, 3JH,H = 5.5 Hz, 4H, H-9),3.28 (s, 6H, H-11), 2.63 (t, 3JH,H = 8.0 Hz, 4H, H-2), 2.49 (t,3JH,H = 7.1 Hz, 4H, H-6), 2.38 (t,3JH,H = 7.5 Hz, 4H, H-7), 1.89(m, 2H, H-1) ppm. 13C NMR: δ = 180.1 (C-5), 176.9 (C-4),171.8 (C-8), 71.1 (C-10), 57.9 (C-11), 56.4 (C-3), 38.9 (C-9),31.9 (C-2), 31.6 (C-6), 31.2 (C-7), 16.0 (C-1) ppm. 15N NMR:δ = −53.8 (NH3), 88.2 (NH-amide) ppm. 195Pt NMR: δ =3561 ppm. IR (ATR): 3531 br, 3224 br (νN–H); 2945 w; 1629 s,1551 m (νCvO); 1338 s, 1257 m, 1210 w, 1121 w cm−1.
(OC-6-33)-Diammine(cyclobutane-1,1-dicarboxylato)bis((4-cyclopentylamino)-4-oxobutanoato)platinum(IV) (3h). CDI(210 mg, 1.295 mmol) in DMF (8 mL), 3 (392 mg,0.648 mmol) in DMF (8 mL), cyclopentylamine (0.14 ml,1.417 mmol) in DMF (3 mL). The crude product was purifiedby column chromatography (EtOAc/MeOH, 3 : 1). The yellowpowder, obtained via suspension in EtOAc, was recrystallizedfrom MeOH, precipitated with EtOAc, filtered off, washed withEt2O and dried under vacuum. A pale yellow to almost whitepowder was obtained. Yield: 184 mg, 38%. Mr = 739.67 gmol−1. Elemental analysis, found: C 37.71, H 5.19, N 7.40.Calcd for C24H40N4O10Pt·H2O: C 38.04, H 5.59, N 7.39.ESI-MS: m/z 761.9 [M + Na+]+, 739.0 [M + H+]+, 738.1 [M −H+]−, 774.7 [M + Cl−]−. 1H NMR: δ = 7.77 (d, 3JH,H = 6.7 Hz,2H, CONH), 6.73 (m, 6H, NH3), 4.07 (m, 2H, H-9), 2.62 (t, 3JH,H = 7.9 Hz, 4H, H-2), 2.46 (t, 3JH,H = 7.2 Hz, 4H, H-6), 2.32 (t,3JH,H = 7.4 Hz, 4H, H-7), 1.89 (m, 2H, H-1), 1.82 (m, 4H,H-10), 1.65 (m, 4H, H-11), 1.52 (m, 4H, H-11), 1.43 (m, 4H,H-10) ppm. 13C NMR: δ = 180.2 (C-5), 176.8 (C-4), 171.1(C-8), 56.3 (C-3), 50.8 (C-9), 32.5 (C-10), 31.8 (C-2), 31.6(C-7), 31.3 (C-6), 23.6 (C-11), 16.0 (C-1) ppm. 15N NMR: δ =−54.3 (NH3), 105.8 (NH-amide) ppm. 195Pt NMR: δ =3562 ppm. IR (ATR): 3260 br, 3086 br (νN–H); 2952 m, 2870 w;1622 s, 1544 m (νCvO); 1332 s, 1252 m, 1207 m, 1097 w cm−1.
(OC-6-33)-Diammine(cyclobutane-1,1-dicarboxylato)bis-((4-cyclohexylamino)-4-oxobutanoato)platinum(IV) (3i). CDI(190.7 mg, 1.176 mmol) in DMF (6 mL), 3 (356 mg,0.588 mmol) in DMF (8 mL), cyclohexylamine (0.15 ml,1.311 mmol) in DMF (2 mL). The crude product was purifiedby column chromatography (EtOAc/IPA, 2 : 1), then isolated viasuspension in EtOAc, filtration, washing with EtOAc and Et2Oand drying under vacuum to obtain a white powder. Yield:162 mg, 36%. Mr = 767.7 g mol−1. Elemental analysis, found:C 40.29, H 5.50, N 7.22. Calcd for C26H44N4O10Pt: C 40.68,H 5.78, N 7.30. ESI-MS: m/z 789.1 [M + Na+]+, 768.0 [M +H+]+, 766.2 [M − H+]−. 1H NMR: δ = 7.66 (d, 3JH,H = 7.6 Hz,2H, CONH), 6.73 (m, 6H, NH3), 3.59 (m, 2H, H-9), 2.62 (t, 3JH,H = 7.9 Hz, 4H, H-2), 2.47 (t, 3JH,H = 7.7 Hz, 4H, H-6), 2.33 (t,3JH,H = 7.2 Hz, 4H, H-7), 1.89 (m, 2H, H-1), 1.79 (m, 4H,H-10), 1.69 (m, 4H, H-11), 1.56 (m, 2H, H-12), 1.27 (m, 4H,H-11), 1.18 (m, 4H, H-10), 1.16 (m, 2H, H-12) ppm. 13C NMR:
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δ = 180.1 (C-5), 176.8 (C-4), 170.6 (C-8), 56.3 (C-3), 47.9(C-9), 32.7 (C-10), 31.8 (C-2), 31.7 (C-7), 31.3 (C-6), 25.6(C-12), 24.9 (C-11), 16.0 (C-1) ppm. 15N NMR: δ = −54.3(NH3), 108.1 (NH-amide) ppm. 195Pt NMR: δ = 3562 ppm. IR(ATR): 3319 br, 3272 br (νN–H); 2929 m, 2854 w; 1658 s, 1639s, 1538 m (νCvO); 1442 w, 1330 s, 1256 w, 1209 w cm−1.
(OC-6-33)-Diamminebis((4-benzylamino)-4-oxobutanoato)-(cyclobutane-1,1-dicarboxylato)platinum(IV) (3j). CDI (175 mg,1.079 mmol) in DMF (7 mL), 3 (322 mg, 0.532 mmol) in DMF(8 mL), benzylamine (145 μl, 1.329 mmol) in DMF (5 mL). Thecrude product was purified by column chromatography (EtOAc/MeOH, 4 : 1) and then isolated via suspending in EtOAc. Theobtained substance was recrystallized from MeOH, and precipi-tated with the help of acetone and ethyl acetate, filtered off,washed with diisopropyl ether and dried under vacuum to obtaina white powder. Yield: 140 mg, 34%. Mr = 783.7 g mol−1.Elemental analysis, found: C 42.60, H 4.67, N 6.80. Calcd forC28H36N4O10Pt: C 42.91, H 4.63, N 7.15. ESI-MS: m/z 805.9[M + Na+]+, 783.0 [M + H+]+, 781.9 [M − H+]−, 818.6 [M +Cl−]−. 1H NMR: δ = 8.28 (t, 3JH,H = 5.5 Hz, 2H, CONH), 7.34(m, 4H, H-12), 7.31 (m, 4H, H-11), 7.25 (m, 2H, H-13), 6.75(m, 6H, NH3), 4.38 (d, 3JH,H = 5.9 Hz, 4H, H-9), 2.64 (t, 3JH,H =8.0 Hz, 4H, H-2), 2.54 (t, 3JH,H = 7.1 Hz, 4H, H-6), 2.44 (t,3JH,H = 7.3 Hz, 4H, H-7), 1.89 (m, 2H, H-1) ppm. 13C NMR:δ = 180.0 (C-5), 176.8 (C-4), 171.7 (C-8), 140.0 (C-10), 128.3(C-12) 127.4 (C-11), 126.8 (C-13), 56.3 (C-3), 42.6 (C-9), 31.8(C-2), 31.6 (C-7), 31.1 (C-6), 16.0 (C-1) ppm. 15N NMR: δ =−54.5 (NH3), 93.6 (NH-amide) ppm. 195Pt NMR: δ =3562 ppm. IR (ATR): 3258 br, 3076 br (νN–H); 2954 w; 1626 s,1545 m (νCvO); 1420 w, 1339 s, 1245 m, 1211 w cm−1.
(OC-6-33)-Diammine(cyclobutane-1,1-dicarboxylato)bis-((5-propylamino)-5-oxopentanoato)platinum(IV) (4f). CDI (164 mg,1.011 mmol) in DMF (8 mL), 4 (289 mg, 0.456 mmol) in DMF(5 mL), propylamine (85 μl, 1.021 mmol) in DMF (3 mL). Thecrude product was purified twice by column chromatography(EtOAc/MeOH, 1 : 1, followed by EtOAc/MeOH, 3 : 1), thenisolated from an EtOAc suspension and washed with a coldmixture of EtOAc and Et2O. The white powder obtained wasdried under vacuum. Yield: 60 mg, 18%. Mr = 715.65 g mol−1.Elemental analysis, found: C 36.58, H 5.34, N 7.58. Calcd forC22H40N4O10Pt: C 36.92, H 5.63, N 7.83. ESI-MS: m/z 737.1[M + Na+]+, 716.1 [M + H+]+, 715.4 [M − H+]−. 1H NMR: δ =7.65 (bs, 2H, CONH), 6.78 (m, 6H, NH3), 3.09 (m, 4H, H-9),2.62 (t, 3JH,H = 8.0 Hz, 4H, H-2), 2.23 (t, 3JH,H = 7.5 Hz, 4H,H-6), 2.14 (t, 3JH,H = 7.4 Hz, 4H, H-7), 1.89 (m, 2H, H-1), 1.74(m, 4H, H-7′), 1.46 (m, 4H, H-10), 0.87 (t, 3JH,H = 7.4 Hz, 6H,H-11) ppm. 13C NMR: δ = 180.3 (C-5), 176.9 (C-4), 172.0(C-8), 56.4 (C-3), 40.7 (C-9), 35.1 (C-7), 35.0 (C-6), 31.8 (C-2),22.8 (C-10), 22.0 (C-7′), 16.0 (C-1), 11.1 (C-11) ppm. 15NNMR: δ = −54.4 (NH3), 93.6 (NH-amide) ppm. 195Pt NMR: δ =3555 ppm. IR (ATR): 3250 br, 3088 br (νN–H); 2960 w; 1637 s,1629 s, 1560 m (νCvO); 1458 w, 1336 s, 1233 m cm−1.
(OC-6-33)-Diammine(cyclobutane-1,1-dicarboxylato)bis-(3-methyl-(5-propylamino)-5-oxopentanoato)platinum(IV) (5f).CDI (222 mg, 1.3691 mmol) in DMF (8 mL), 5 (453 mg,0.6848 mmol) in DMF (10 mL), propylamine (0.15 ml,1.8170 mmol) in DMF (3 mL). The crude product was purified
by column chromatography (EtOAc/MeOH/IPA, 3 : 1 : 1, fol-lowed by EtOAc/MeOH, 3 : 1 for the second column), then iso-lated from an EtOAc suspension and washed with a cold mixtureof EtOAc and Et2O. The white powder obtained was dried undervacuum. Yield: 252 mg, 49%. Mr = 743.70 g mol−1. Elementalanalysis, found: C 38.10, H 5.78, N 7.51. Calcd forC24H44N4O10Pt·0.5(H2O): C 38.30, H 6.03, N 7.44. ESI-MS:m/z 782.0 [M + K+]+, 766.0 [M + Na+]+, 743.1 [M + H+]+,742.2 [M − H+]−. 1H NMR: δ = 7.68 (bs, 2H, CONH), 6.81 (m,6H, NH3), 3.10 (m, 4H, H-9), 2.63 (t, 3JH,H = 7.9 Hz, 4H, H-2),2.29 (m, 2H, H-7′), 2.25 (m, 2H, H-6), 2.17 (m, 2H, H-7), 2.10(m, 2H, H-6), 2.00 (m, 2H, H-7), 1.90 (m, 2H, H-1), 1.47 (m,4H, H-10), 0.90 (d, 3JH,H = 4.7 Hz, 6H, H-7′′), 0.88 (t, 3JH,H =7.4 Hz, 6H, H-11) ppm. 13C NMR: δ = 180.0 (C-5), 177.0(C-4), 171.5 (C-8), 56.4 (C-3), 42.9 (C-7), 42.7 (C-6), 40.7(C-9), 32.0 (C-2), 28.6 (C-7′), 22.8 (C-10), 19.6 (C-7′′), 16.0(C-1), 11.2 (C-11) ppm. 15N NMR: δ = −54.3 (NH3), 96.7 (NH-amide) ppm. 195Pt NMR: δ = 3560 ppm. IR (ATR): 3259 br,3084 br (νN–H); 2962 m, 2875 w; 1624 s, 1551 m(νCvO); 1458w, 1344 m, 1304 m, 1221 w, 1098 w cm−1.
(OC-6-33)-Diammine(cyclobutane-1,1-dicarboxylato)bis-(3,3-dimethyl-(5-propylamino)-5-oxopentanoato)platinum(IV) (6f).CDI (152 mg, 0.9343 mmol) in DMF (10 mL), 6 (263 mg,0.3810 mmol) in DMF (8 mL), propylamine (0.1 ml,1.2112 mmol) in DMF (2 mL). The crude product was purifiedby column chromatography (EtOAc/MeOH/IPA, 4 : 1 : 1), thenisolated from an EtOAc suspension and recrystallized fromMeOH, suspended in EtOAc/Et2O mixture, filtered off, washedwith Et2O and dried under vacuum to obtain a white powder.Yield: 98 mg, 33%. Mr = 771.76 g mol−1. Elemental analysis,found: C 40.08, H 5.99, N 7.41. Calcd for C26H48N4O10Pt:C 40.46, H 6.27, N 7.26. ESI-MS: m/z 810.0 [M + K+]+, 794.0[M + Na+]+, 771.2 [M + H+]+, 770.0 [M − H+]−, 806.9 [M +Cl−]−. 1H NMR: δ = 7.60 (t, 3JH,H = 5.5 Hz, 2H, CONH), 6.83(m, 6H, NH3), 3.09 (m, 4H, H-9), 2.63 (t, 3JH,H = 8.0 Hz, 4H,H-2), 2.28 (s, 4H, H-6), 2.16 (s, 4H, H-7), 1.90 (m, 2H, H-1),1.46 (m, 4H, H-10), 1.03 (s, 12H, C-7′′), 0.88 (t, 3JH,H = 7.4 Hz,6H, H-11) ppm. 13C NMR: δ = 179.7 (C-5), 176.9 (C-4), 170.9(C-8), 56.3 (C-3), 47.3 (C-6), 47.2 (C-7), 40.6 (C-9), 32.9(C-7′), 31.9 (C-2), 27.5 (C-7′′), 22.8 (C-10), 16.0 (C-1), 11.2(C-11) ppm. 15N NMR: δ = −55.5 (NH3), 98.7 (NH-amide)ppm. 195Pt NMR: δ = 3568 ppm. IR (ATR): 3266 br, 3075 br(νN–H); 2964 m, 2933 m; 1626 s, 1560 m (νCvO); 1472 w,1345 m, 1333 m, 1241 w cm−1.
Determination of lipophilicity
Lipophilicity of the new compounds was determined as chroma-tographic retention factors (namely log k30 and log kw) usingreversed-phased HPLC.
The analysis was performed on a Dionex Summit system con-trolled by the Dionex Chromeleon 6.60 software. The sampleswere prepared by dissolving (with the help of ultrasonic) around1.5 mg of each complex in a H2O/MeOH mixture, followed byfiltration through a 0.2 μm Nylon filter. The chromatographicconditions were as follows: an Agilent ZORBAX Bonus-RPcolumn (4.6 mm × 250 mm); injection volume: 25 μl; flow rate:1 ml min−1; isocratic elution; temperature of the column: 25 °C;
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UV-vis detection set up at 210 nm; uracil was used as an internalreference to determine the column dead-time (t0); mobile phasescontaining different percentages of 0.1% TFAwater solution andMeOH (the MeOH fraction ranged from 60% for the most lipo-philic compounds to 10% for the most hydrophilic compounds);chromatograms for each complex were run with at least threedifferent mobile phase compositions and at least two times.
The capacity factors k = (tR − t0)/t0 (tR is the retention time ofthe species analyzed and t0 is the retention time of uracil) of theinvestigated compounds were calculated for all eluant compo-sitions. The partition between the lipophilic stationary phase andwater (0% MeOH) was determined by extrapolation, using thelinear Soczewinski–Snyder relationship43,44 between log k andthe concentration of the organic modifier in the mobile phase:
log k ¼ log kw � Sφ;
where log k is the capacity factor in the specific mobile phasecomposition, φ is the volume fraction of MeOH in the eluant,S is a constant for a given solute and a given HPLC system andlog kw corresponds to log k in pure water (buffer).
Electrochemical experiments
Cyclic voltammograms were measured in a three-electrode cellusing a 2.0 mm diameter glassy carbon disc working electrode, aplatinum auxiliary electrode, and a Ag/Ag+ reference electrodecontaining 0.10 M AgNO3, the potential of which was correctedusing an internal standard of ferrocenium/ferrocene. Measure-ments were performed at room temperature using an EG & GPARC 273A potentiostat/galvanostat. Deaeration of solutionswas accomplished by passing a stream of argon through the solu-tion for 5 min prior to the measurement and then maintaining ablanket atmosphere of argon over the solution during themeasurement. The potentials were measured at a scan rate of100 mV s−1 in 0.15 M [n-Bu4N][BF4]/DMF, using [Fe(η5-C5H5)2] (E1
2= +0.72 V vs. NHE)45 as the internal standard, and
are quoted relative to the normal hydrogen electrode (NHE).
Incubation with ascorbic acid
Reduction of complex 3f and its close analogue featuringethane-1,2-diamine and two chlorido ligands in the equatorialposition (M1) by ascorbic acid was monitored by 1H NMR spec-troscopy at ambient temperature. 1 mM solutions of the com-pounds were prepared in 50 mM phosphate buffer (in D2O, pD =7.4) and 1H NMR spectra were measured from time to time overa period of 24 h in order to judge the stability of the complexes;changes in the NMR spectra could not be seen. Then, ascorbicacid (25 mM) was added and 1H NMR spectra were recorded for24 h in the case of M1 and during three weeks for complex 3f.The reduction was monitored by following the decrease of inten-sity of the CH2 signal of the ethane-1,2-diamine ligand of M1resonating at 2.8 ppm. In the case of complex 3f, the increase ofintensity of CH2 protons H2 at 2.8 ppm deriving from the cyclo-butanedicarboxylato ligand of the reduced platinum(II) specieswas monitored. In both cases, integration of the signals was per-formed relative to the signals of the terminal methyl groups ofthe propylamine moieties.
Cell lines and culture conditions
CH1 (ovarian carcinoma, human) cells were a gift from LloydR. Kelland (CRC Centre for Cancer Therapeutics, Institute ofCancer Research, Sutton, U.K.). A549 (non-small cell lungcancer, human) and SW480 (colon carcinoma, human) cellswere kindly provided by Brigitte Marian (Institute of CancerResearch, Department of Medicine I, Medical University ofVienna, Austria), and SK-OV-3 (ovarian carcinoma, human)cells by Evelyn Dittrich (General Hospital, Medical Universityof Vienna, Austria). Cells were grown in 75 cm2 culture flasks(Iwaki/Asahi Technoglass) as adherent monolayer cultures inMinimal Essential Medium (MEM) supplemented with 10%heat-inactivated fetal bovine serum, 1 mM sodium pyruvate,4 mM L-glutamine and 1% non-essential amino acids (from100× ready-to-use stock) (all purchased from Sigma-Aldrich)without antibiotics. Cultures were maintained at 37 °C in ahumidified atmosphere containing 5% CO2 and 95% air.
Cytotoxicity tests in cancer cell lines
Cytotoxicity in the cell lines mentioned above was determinedby the colorimetric MTT assay (MTT = 3-(4,5-dimethyl-2-thia-zolyl)-2,5-diphenyl-2H-tetrazolium bromide, purchased fromFluka). Cells were harvested from culture flasks by trypsinizationand seeded in 100 μL aliquots in MEM (see above) into 96-wellmicroculture plates (Iwaki/Asahi Technoglass) in the followingdensities to ensure exponential growth of untreated controlsthroughout the experiment: 1.5 × 103 (CH1), 3.5 × 103
(SK-OV-3), 4.0 × 103 (A549), and 2.5 × 103 (SW480) viablecells per well. Cells were allowed to settle and resume exponen-tial growth in a drug-free medium for 24 h, followed by theaddition of dilutions of the test compounds in 100 μL per well ofthe same medium. Only for the less water soluble compound 3e,a stock solution in DMSO/water was prepared (the DMSOcontent in the cytotoxicity assay did not exceed 0.5%). Aftercontinuous exposure for 96 h, the medium was replaced by a100 μL per well RPMI 1640 medium (supplemented with 10%heat-inactivated fetal bovine serum and 4 mM L-glutamine) plusa 20 μL per well solution of MTT in phosphate-buffered saline(5 mg mL−1) (all purchased from Sigma-Aldrich). After incu-bation for 4 h, medium/MTT mixtures were removed, and theformazan product formed by viable cells was dissolved inDMSO (150 μL per well). Optical densities at 550 nm weremeasured with a microplate reader (Tecan Spectra Classic), usinga reference wavelength of 690 nm to correct for unspecificabsorption. The quantity of viable cells was expressed as a per-centage of untreated controls, and 50% inhibitory concentrations(IC50) were calculated from concentration–effect curves byinterpolation. Evaluation is based on means from three indepen-dent experiments, each comprising six replicates per concen-tration level.
Acknowledgements
H. V. is thankful for financial support from the University ofVienna within the doctoral program Initiativkolleg FunctionalMolecules IKI041-N. The authors are indebted to the FFG –
Austrian Research Promotion Agency, the Austrian Council for
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Research and Technology Development, the FWF (AustrianScience Fund, P20683-N19) and COST D39.
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S1
Electronic Supporting Information
Novel tetracarboxylatoplatinum(IV) complexes as carboplatin prodrugs
Hristo P. Varbanov, Seied M. Valiahdi, Christian R. Kowol, Michael A. Jakupec, Markus
Galanski*, and Bernhard K. Keppler*
University of Vienna, Institute of Inorganic Chemistry, Waehringer Strasse 42, A-1090
Vienna, Austria
Contents
page S2 Figure S1 with the ORTEP diagram of 4.
page S3 Figure S2 with concentration–effect curves of investigated compounds
in CH1 cells.
page S4 Figure S3 with chemical structures of complexes 3f and M1
page S4 Figure S4 with the time dependent reduction of M1 and 3f in the
presence of ascorbic acid.
page S5 Figure S5 with 1H NMR spectra of complex M1 after addition of
ascorbic acid.
page S6 Figure S6 with 1H NMR spectra of complex 3f after addition of
ascorbic acid.
page S6 Table S1 with the comparison of redox potentials, halve life times of
reduction by ascorbic acid and cytotoxicity for complexes M1 and 3f.
tronic Supplementary Material (ESI) for Dalton Transactionsjournal is © The Royal Society of Chemistry 2012
98
S2
Fig. S1. ORTEP diagram of 4 displaying thermal ellipsoids at 55% probability level.
Electronic Supplementary Material (ESI) for Dalton TransactionsThis journal is © The Royal Society of Chemistry 2012
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Fig. S2. Concentration–effect curves (means ± standard deviations) of investigated compounds in CH1 cells (MTT assay, exposure time 96 h).
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S4
Pt
O
NH3
NH3
O
O
O
NH
O
O
O
O
O
HN
O Pt
O
Cl
Cl
H2N
NH2
O
NH
O
O
O
HN
O
3f M1
Fig. S3. Chemical structures of complexes 3f and M1
0,00
20,00
40,00
60,00
80,00
100,00
120,00
0 5 10 15 20 25
t (hours)
%P
t(IV
)
0
20
40
60
80
100
120
0 5 10 15 20 25
t (days)
%P
t(IV
)
Fig. S4. Time dependent reduction of M1 (top) and 3f (bottom) in the presence of ascorbic
acid; ambient temperature, pD =7.4, 1 mM complex, 50 mM phosphate buffer, 25 mM
ascorbic acid.
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S5
ppm (t1)1.001.502.002.503.00
ppm (t1)1.001.502.002.503.00
ppm (t1)1.001.502.002.503.00
Fig. S5. 1H NMR spectra of complex M1 after addition of ascorbic acid (top – immediately,
middle – after 5 hours, bottom – after 17 h); ambient temperature, pD =7.4, 1 mM complex,
50 mM phosphate buffer, 25 mM ascorbic acid.
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S6
ppm (t1)1.001.502.002.503.00
ppm (t1)1.001.502.002.503.00
ppm (t1)1.001.502.002.503.00
Fig. S6. 1H NMR spectra of complex 3f after addition of ascorbic acid (top – immediately,
middle – after 3 days, bottom – after 23 days); ambient temperature, pD =7.4, 1 mM complex,
50 mM phosphate buffer, 25 mM ascorbic acid
Table S1. Comparison of redox potentials, halve life times of reduction by ascorbic acid and
cytotoxicity for complexes M1 and 3f.
compound Ep (V) t1/2 IC50 (CH1), μM IC50 (SW480), μM
M1 -0.60 5 h 2.3±1.1a 31±15a
3f -0.68 21 d 44 ± 8 >500 a data, taken from ref. 1
1 M.R. Reithofer, S.M. Valiahdi, M.A. Jakupec, V.B. Arion, A. Egger, M. Galanski, B.K. Keppler, J. Med. Chem., 2007, 50, 6692–6699.
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3. Theoretical Investigations and Density Functional Theory Based
Quantitative Structure–Activity Relationships Model for Novel
Cytotoxic Platinum(IV) Complexes.
H.P. Varbanov, M.A. Jakupec, A. Roller, F. Jensen, M. Galanski, B.K. Keppler, J. Med.
Chem., 2013, 56, 330-344.
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106
Theoretical Investigations and Density Functional Theory BasedQuantitative Structure−Activity Relationships Model for NovelCytotoxic Platinum(IV) ComplexesHristo P. Varbanov,† Michael A. Jakupec,† Alexander Roller,† Frank Jensen,*,‡ Markus Galanski,*,†
and Bernhard K. Keppler†
†Institute of Inorganic Chemistry, University of Vienna, Wahringer Strasse 42, A-1090 Vienna, Austria‡Department of Chemistry, University of Aarhus, Langelandgade 140, 8000 Aarhus C, Denmark
*S Supporting Information
ABSTRACT: Octahedral platinum(IV) complexes are promising candidates in thefight against cancer. In order to rationalize the further development of this class ofcompounds, detailed studies on their mechanisms of action, toxicity, and resistancemust be provided and structure−activity relationships must be drawn. Herein, we reporton theoretical and QSAR investigations of a series of 53 novel bis-, tris-, and tetrakis-(carboxylato)platinum(IV) complexes, synthesized and tested for cytotoxicity in ourlaboratories. The hybrid DFT functional wb97x was used for optimization of the structuregeometry and calculation of the descriptors. Reliable and robust QSAR models with goodexplanatory and predictive properties were obtained for both the cisplatin sensitive cell lineCH1 and the intrinsically cisplatin resistant cell line SW480, with a set of four descriptors.
■ INTRODUCTION
Platinum complexes are among leading drugs in anticancer che-motherapy. Since the discovery of the cytotoxic effect of cisplatinand its Food and Drug Administration (FDA) approval in 1978,seven other Pt(II) compounds were introduced in clinics world-wide (carboplatin andoxaliplatin) or in selected countries (nedaplatin,lobaplatin, heptaplatin, miriplatin, and dicycloplatin).1−3 Approx-imately 30 more Pt(II) and Pt(IV) complexes have been or are inclinical trials at different stages.1 Despite the great medicalsuccess of platinum-based cytostatics, there are some majordrawbacks that restrict their usage, mainly severe dose-limitingside effects, intrinsic or/and acquired resistance, and the uncom-fortable and cost intensive way of administration (iv infusion).Thousands of metal compounds have been synthesized and in-vestigated during the past decades with the aim of breaking theselimitations. Nevertheless, in order to design ametal-based drug withimproved pharmacological profile, details of the mechanism ofaction, toxicity, and resistance have to be studied4 and structure−activity relationships have to be drawn. It is generally accepted thatsquare planar platinum(II) complexes are acting like prodrugs,containing two carrier ligands and two leaving groups. The twoleaving groups are exchanged in the cell, forming reactive aquaspecies capable of forming DNA adducts responsible for the cyto-toxic effects of the compounds (Figure 1). Octahedral Pt(IV) com-plexes also possess antimalignant properties and can act as prodrugsfor Pt(II) agents (reduction in vivo to the corresponding Pt(II)counterparts).The first comprehensive SAR study of cytotoxic metal com-
plexes was reported by Cleare and Hoeschele in 1973, where a
wide variety of Pt(II) compounds was investigated for its anti-tumor activity in a sarcoma 180 mouse model.5 Results fromvariation of carrier ligands, leaving groups, geometry, and chargeand some physicochemical parameters like solubility and kineticsof hydrolysis affecting the antimalignant properties of cisplatinanalogues were studied. The authors found that the cis geometryand neutral charge of the complexes, chloride or dicarboxylates asleaving groups, and primary amines as carrier ligands are crucialfor the biological activity within the series studied. Today, dif-ferent compound classes are known, violating the classical SARset up by Cleare and Hoeschele, as for example complexes withtrans geometry featuring high cytotoxicity.6 Theoretical studyattempts and a quantitative structure−activity relationship (QSAR)model for the anticancer activity of 26 Pt(II) complexes in vivo inmice models was reported in 1982.7 Nevertheless, QSAR analysisresults based on in vitro cytotoxicity of Pt(II) compounds indifferent cell lines were first published 23 years later.8 Reliablemodels with good predictive strength, based on four moleculardescriptors (chosen from 197), were obtained for a series of 16Pt(II) complexes, including the clinically established drugs cisplatin,carboplatin, and oxaliplatin. The results confirmed the structure−activity relationships (SAR) reported byCleare andHoechele. Later,Sarmah and Deka reported QSAR and quantitative structure−properties relationship (QSPR) models for several platinum com-plexes, using density functional theory (DFT) and MM deriveddescriptors.9 The authors showed that DFT and molecular
Received: November 7, 2012Published: December 10, 2012
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mechanics (MM+) methods could be used successfully in theprediction of lipophilicity and cytotoxicity of platinum com-pounds. Furthermore, the usage of solvent models for calculationof the descriptors gave better results than those obtained in thegas phase.Asmentioned above, Pt(IV) complexes act as prodrugs of their
Pt(II) counterparts and represent an important part of recentmetal-based anticancer research. Their geometries and phys-icochemical features (octahedral coordination sphere with a max-imum of six ligands, kinetic inertness in ligand-exchange reactions,reduction under hypoxic conditions, etc.) present advantages in fine-tuning of the pharmacological profile, providing the possibility fororal administration, targeted therapy, reduced side effects, etc.10
As summarized in Figure 1, there are more parameters (incomparison with platinum(II) complexes), which should betaken into account when designing a Pt(IV) based drug. SomeSARs based on a small set of Pt(IV) complexes have been estab-lished during the past decade.11 It was shown that cytotoxicity ofthe compounds is dependent on their redox potential andlipophilicity and that these parameters have optimal values whenthe axial ligands are carboxylates.12 However, it was found re-cently that redox potential does not always correlate with the rateof reduction and that the equatorial ligands can also play a crucialrole.13,14 Moreover, reduction of Pt(IV) complexes is not alwaysaccompanied by release of the axial ligands; in some rare cases amore complicated picture can be observed.15,16
cis-Diam(m)inebis(carboxylato)dichloridoplatinum(IV) andcis-diam(m)inetetrakis(carboxylato)platinum(IV) complexeswith cytotoxicity ranging from low nanomolar to high micromolarIC50 values have recently been reported from our group (seeFigure 2).14,17−20 It was found that cytotoxicity in general increaseswith increasing lipophilicity of the axial ligands, but this effect ismuchmore pronounced in the diam(m)inedichloridobis(carboxylato)
complex series; moreover, compounds featuring amide moietiesin the axial ligands are less effective than expected from their log Pvalues.20,21 The tetracarboxylato complexes have shown inprinciple a lower cytotoxic potency and a different redox kineticbehavior.14 In order to find quantitative explanations of thephenomena and to rationalize the further development of anti-malignant Pt(IV) complexes, we enlarged the series by includingthree diamminetris(carboxylato)platinum(IV) complexes, pro-drugs of nedaplatin, and performed a QSAR study based on DFTcalculated and constitutional molecular descriptors. Moreover,with the help of the calculations, we tried to better understandthe redox behavior of the complexes in the series to explain theexperimental data and to group them in subseries.Up to now, there is only one report of a QSAR study for Pt(IV)
complexes;22 models based on the cytotoxicity of 23 compoundsin two tumor cell lines were developed, using experimentallydetermined (log Po/w and Ep) and theoretical descriptors. Later,the authors suggested QSPR models able to predict the lipo-philicity and the redox potential of Pt(IV) complexes, using (aslightly broadened) series from the QSAR study. The semi-empirical method PM6was used for optimization of the structuresand calculation of the descriptors.23 Total and polar surface area,orbital energies, atomic charges, and dipole moments were foundto be significant descriptors.To the best of our knowledge, there is still no QSAR study on
Pt(IV) complexes based on DFT derived descriptors in theliterature. Herein, we report theoretical and QSAR investigationson 53 novel Pt(IV) complexes (listed in Figure 2), synthesizedand tested in our laboratories, using the hybrid DFT functionalwb97x for optimization of the structure geometry and calculationof the descriptors. MLR, PCA, and simulated annealing wereemployed for the development of the statistical models.
Figure 1. Scheme of the mechanism of action of platinum-based cytostatics.
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■ RESULTS AND DISCUSSIONSynthesis and Characterization. The entire set of 53
Pt(IV) complexes, which are an object of this study, is presentedin Figure 2. On the basis of the equatorial ligands, the com-pounds can be divided into six subseries, namely, derivatives ofcisplatin (1−5), its ethylenediamine analogue (6−20), its bis-(ethylamine) analogue (21−26), carboplatin (27−47), neda-platin (48−50), and oxaliplatin (51−53). The axial ligands arerepresented by dicarboxylato chains, containing different spacersbetween the two carbonyl groups and diverse terminal moieties
such as esters, amides, or free carboxylic acids. Synthesis anddetailed characterization of compounds 1−20 and 51−53 aregiven in refs 17, 18, and 19, and those for 21−47 are given in refs14 and 20. Nedaplatin derivatives (48−50) were synthesizedusing analogous procedures. Their detailed characterization isbased on 1H, 13C, 15N, and 195Pt 1D and 2DNMRmeasurements.Interestingly, two different-shaped signals for the NH3 groupscan be observed in the 1H spectra of compounds 48−50: a broadsignal at around 6.1 ppm and amultiplet around 6.6 ppm in whichthe 14N−1H and 195Pt−1H couplings can be seen. The existence oftwo signals in the 1H and 15N spectra can be explained by theunsymmetrical surroundings of the NH3 groups, one is in transposition to carboxylate and the other one to alcoholate.
Cytotoxicity. All complexes (1−53) were tested for in vitrocytotoxicity in comparison with cisplatin, carboplatin, oxaliplatin,and nedaplatin in two human tumor cell lines (CH1 ovariancarcinoma and SW480 colon carcinoma), using the MTT color-imetric assay. The resulting IC50 values are listed in Table 1. Thecell line CH1 is sensitive to the clinically applied platinum drugs,while the second one (SW480) is resistant to them with the ex-ception of oxaliplatin. The set of compounds covers a large rangeof cytotoxicity with nanomolar IC50 values up to 174 μM in thecell line CH1 and from 0.1 μM to negligible activity (>500 μM)in the cell line SW480. In general, the diam(m)inebis(carboxylato)-dichlorido complexes (1−26, subset 1) show higher activity incomparison with the tri- and tetrakis(carboxylato)diam(m)-ine compounds (27−53, subset 2). With increasing the lipo-philicity, complexes with higher cytotoxicity than the clinicallyapplied platinum(II) drugs could be obtained in subset 1, whilethis observation is not valid for the compounds in subset 2. Ingeneral for all complexes in the set, cytotoxicity is dependenton lipophilicity, but this is much more pronounced for thediam(m)inedicarboxylatodichlorido complexes. When the axialligands are compared, terminal ester groups are most favorablefor antiproliferative activity, followed by amide derivatives;compounds featuring terminal carboxylic or hydroxy groups inthe axial chain showed the lowest cytotoxic potency (see Figure 3).In subset 2, cytotoxicity of amide and ester derivatives is comparable.Lack of activity of all compounds from the subset (except foroxaliplatin analogues 51−53 and partially for nedaplatin analogues49−50) in the cisplatin-resistant cell line SW480 can be observed(Table 1).
Crystal Structure. The result of the X-ray diffraction analysisof 7 is shown in Figure S1 in Supporting Information. Thecompound crystallized in the triclinic centrosymmetric spacegroup P1 . The Pt(IV) atom has an octahedral coordinationgeometry with one ethylenediamine and two chlorido ligands inthe equatorial plane and two 4-methoxysuccinates coordinated inaxial positions. The bond lengths and angles are well comparablewith the crystal structure of analogous complex 6 previouslypublished.17 Interestingly, the orientations of the axial ligands incomplex 7 are different from those observed in 6. This is probablydue to dissimilar crystal packing and H-bonding pattern. Ananalogous difference in the conformational behavior was observedin the structures of complexes 1 and 22, whereas the 4-methoxy-succinate ligands in 22 have a straight orientation20 while in 1 onesuccinate is twisted and the other is straight.18
Geometry Optimization. Comparison of geometry param-eters, obtained after the optimization procedure in vacuum, in awater model and from the available X-ray data for compounds 1,6, 7, 22, and 38 is shown in Table S1 (Supporting Information).A good agreement between experiment and calculation could beobserved.
Figure 2. Schematic formulas of the investigated complexes.
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Analysis of the Calculated Physicochemical Param-eters. The dipole moments (μ) vary from 3 to 15 D, implyingthat all the complexes are quite polar compounds (Table S2).Nevertheless, no trend in the alteration of this parameter withinthe investigated compounds could be found.The energies of solvation (Es and Es′) differ from 70 to
160 kJ mol−1 for all compounds. In general the complexes exert-ing carboxylic or amide groups in the axial ligands have highersolvation energies compared with their ester analogues. The dis-tribution of the electron density, based on electrostatic potential(ESP) for compounds of the two subtypes, shows that the mostelectropositive regions in the molecule could be found aroundthe nitrogen donor atoms and the most negative around theoxygen and chlorine atoms (Figure 4).The natural population analysis (NPA) charge at Pt (q(Pt))
differentiates well the two major subtypes (in structure deviationand in cytotoxic activity): bis(carboxylato)dichlorido complexes(subset 1) versus tris- and tetrakis(carboxylato) complexes(subset 2). It also shows some minor discrimination in the twosubtypes, dependent mainly on differences in the equatorialligands. Expectedly, the deviations of the terminal fragments ofthe axial ligands do not affect the charge at the Pt atom. A plot ofthe NPA charge at Pt for the 53 investigated complexes (averagevalues from the calculated conformers of compounds of 2 and 50are used) is shown in Figure 5.
The described circumstances make q(Pt) a good descriptor fora further QSAR model. In Figure 6, a qualitative MO analysis ofthe frontier orbitals, together with their energies for complexes22 and 38, is shown. The low (negative) values ofEHOMO (<−9 eV)and the good correlating high ionization potentials (between 8 and10 eV in vacuum and between 7 and 8.5 eV in water) are a logicalconsequence of the inability of Pt(IV) complexes to act asreductants. However, a clear tendency of the change of these param-eters in the series cannot be observed.The negative values of ELUMO (Table S3, Figure 6) show that
the compounds can act as oxidants and can be reduced relativelyeasily. The nedaplatin derivatives (complexes 48−50) havehigher (close to zero, even slightly positive for complexes 49 and50b) values of ELUMO, which probably is connected with theireventual lower oxidative capability. In general, a clear relationshipbetween the variations of ELUMO with the structures in the seriescould not be found. Interestingly, the values of the electronaffinity in gas phase (between 0.6 and 1.8 eV) and their corre-sponding vertical (between 2 and 3 eV) and adiabatic (around4 eV) redox potentials in water do not correlate with each otheras would be expected. In principle, the small differences observedbetween the values of the adiabatic redox potential in water implythat the investigated compounds can be reduced with relativelyequal effort. The different tendencies for the vertical andadiabatic electron affinity in water also show that the acceptanceof an electron in aqueous media is associated with significant
Table 1. Cytotoxicity of the Investigated Platinum(IV) Complexes in Comparison with the Clinically Applied Platinum(II) Drugsin the CH1 and SW480 Human Cancer Cell Lines
IC50 (μM)a IC50 (μM)a
compd CH1 SW480 compd CH1 SW480
1 19 ± 1 136 ± 16 30 24 ± 5 >5002 0.62 ± 0.32 3.8 ± 1.0 31 8.6 ± 1.7 350 ± 393 28 ± 2 183 ± 28 32 11 ± 6 181 ± 444 12 ± 4 48 ± 4 33 62 ± 26 >5005 1.9 ± 0.2 24 ± 4 34 44 ± 8 >5006 5.5 ± 2.2 95 ± 5 35 15 ± 5 >5007 0.68 ± 0.20 16 ± 1 36 28 ± 2 >5008 0.34 ± 0.11 4.1 ± 0.5 37 31 ± 13 >5009 0.068 ± 0.024 0.63 ± 0.20 38 114 ± 23 >50010 0.018 ± 0.007 0.22 ± 0.08 39 33 ± 13 >50011 24 ± 3 142 ± 23 40 7.7 ± 1.4 >25012 2.3 ± 1.1 31 ± 15 41 89 ± 7 >50013 1.9 ± 0.2 19 ± 9 42 128 ± 48 >50014 32 ± 19 160 ± 10 43 23 ± 9 >50015 1.1 ± 0.2 3.5 ± 0.1 44 49 ± 13 >50016 21 ± 8 90 ± 21 45 125 ± 35 >50017 22 ± 12 43 ± 22 46 22 ± 8 >50018 7.8 ± 1.0 21 ± 5 47 33 ± 4 >50019 0.17 ± 0.05 2.9 ± 1.0 48 21 ± 6 >50020 0.055 ± 0.006 0.96 ± 0.4 49 1.9 ± 0.3 100 ± 621 5.6 ± 1.6 40 ± 12 50 2.1 ± 0.3 161 ± 3322 0.16 ± 0.05 1.0 ± 0.3 51 55 ± 28 44 ± 923 0.061 ± 0.015 0.30 ± 0.05 52 19 ± 5 14 ± 324 0.014 ± 0.002 0.11 ± 0.01 53 11 ± 2 12 ± 525 0.0094 ± 0.0012 0.39 ± 0.07 cisplatin 0.16 ± 0.03 3.50 ± 0.2926 0.75 ± 0.10 6.1 ± 0.6 carboplatin 1.36 ± 0.40 85 ± 2827 171 ± 1 >500 oxaliplatinb 0.33 ± 0.09 0.30 ± 0.0828 32 ± 10 >500 nedaplatin 0.14 ± 0.05 6.3 ± 1.329 28 ± 4 >500
aThe reported 50% inhibitory concentrations are the means ± standard deviations obtained from three independent experiments. bData taken fromref 24.
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changes in the geometry and the energy of the system. It isinteresting to follow how the geometry of the complexes hasbeen changed with the acceptance of one electron. From theNPA charges it can be concluded that the extra electron mainlyresides on the Pt atom (and the coordinated chloride, in the caseof complexes of subset 1), a Pt(III) radical is formed, andexpectedly the largest and important geometry alterations will bein the Pt coordination sphere. In Table 2, a comparison of thebond lengths changes between the neutral and the anion radical(both optimized in water) for complexes 24 and 28 is shown.Table 2 shows that addition of an electron to a complex from
subset 2 results in an elongation of Pt−Oax bonds while Pt−Nand Pt−Oeq in the equatorial sphere remain almost unchanged.These observations are in accordance with the expected reduc-tion and loss of the axial ligands. Contrary to bis(carboxylato)-dichlorido complexes from subset 1 (with the exception of someof the cisplatin analogues, namely, complexes 1, 2c, and 3), theaxial Pt−O bonds have shifted insignificantly but the equatorialPt−Cl and trans standing Pt−N have been elongated by more
than 0.3 Å. In principle, the last findings correlate well with theshape of the LUMO orbitals which, for complexes from subset 2,are mainly situated around the axial ligands while for those fromsubset 1 they could be found in the square-planar sphere aroundplatinum (Figure 6). Consequently, a different mechanism ofreduction between the complexes of the two main subtypes isexpected.
Reduction Model Studies. In order to gain a deeper insightinto the mechanism of reduction of Pt(IV) prodrugs, furtherinvestigations based on two simple model systems, namely,(OC-6-33)-bis(acetato)diamminedichloridoplatinum(IV)(M1), representing complexes of subset 1, and (OC-6-33)-bis(acetato)diamminemalonatoplatinum(IV) (M2), represent-ing complexes of subset 2 (Figure S2, Supporting Information),were performed. Applying again the most prominent hypothesisfor the reduction pathway of Pt(IV) complexes featuring axialcarboxylato ligands, which is an outer sphere reduction going viaa Pt(III) intermediate,15 the structures of M1, M2, and theiranalogous monoanionic Pt(III) radicals were optimized in awater model. The same was done for the respective intermediatesof reduction, the pentacoordinate complex after cleavage (ionicor radical) of one ligand (acetate or chloride). The energy of thecleaved acetate and chloride in water was also calculated. Thepossible ligand dissociation reactions after one-electron reduc-tion are presented schematically in Figure 7. The dissociationenergies, calculated for possible ionic or radical cleavage of aligand (chloride or acetate) from the neutral Pt(IV) complexes orfrom their anion radicals, are listed in Table 3.
Figure 3. Comparative diagram of the cytotoxicity (IC50 values, logarithmic scale) of some Pt(IV) complexes from the series in the CH1 cell line,depending on their equatorial ligands (y axis) and the terminal moieties of the axial ligands (x axis), and the clinically approved Pt(II) drugs: cisplatin,carboplatin, oxaliplatin, and nedaplatin.
Figure 4. ESP color mapped electron density for complexes 22 (left)and 36 (right).
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As presented in Table 3, reasonable dissociation energies areobserved only when a ligand is cleaved from a Pt(IV) complex,which has already accepted an electron (and has been trans-formed into a Pt(III) radical). From the obtained values, it can be
concluded that cleavage of equatorially bound chloride fromthe ionized complex M1 requires less energy than dissociationof axial acetate. Furthermore, the dissociation of an axialcarboxylato ligand appears to happen more easily in the case of
Figure 5. NPA charge at the Pt atom (in au), calculated for complexes 1−53. A scheme of the coordination sphere of subsets 1 and 2 is presented onthe right.
Figure 6. Frontier orbitals (with their energies) of complexes 22 (top) and 38 (bottom).
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bis(carboxylato)dichlorido complexes (like M1) in comparisonto tetracarboxylato species (likeM2). The last findings correlatewith the experimental data from electrochemical experiments,where it was found that bis(carboxylato)dichlorido(ethane-1,2-diammine)platinum(IV) complexes (6−20) have similar butslightly higher redox potentials (approximately −0.6 V vs NHE)than the corresponding carboplatin analogues (27−47) (approx-imately−0.7 V vsNHE).14,21Nevertheless, the electron affinity (theenergy released after the attachment of an electron to a neutralcomplex) is much higher than the obtained dissociation energies:380.2 kJ/mol for M1 and 392.0 kJ/mol for M2. For this reason,reduction of both complexes with dissociation of acetate or chlorideis possible, which is in agreement withGibson’s observation formorethan one product of reduction of diacetatodiam(m)inedichlorido-platinum(IV) complexes25 as well as with the nonaxial ligand lossreduction recently reported by Hambley/Gibson13 and Cullinane.26
The thermodynamically comparable redox properties (intheory and experimentally) for both types of compounds weredifferent with respect to their kinetic behavior. Compounds ofsubset 1 were reduced by ascorbic acid much more quickly thanthose from subset 2.14 In order to learnmore about the kinetics ofreduction, modeling of the energy change as a function of elonga-tion of the Pt−acetate bond (in both models) or Pt−Cl (inM1)was performed. Unfortunately, the attempts to find an energybarrier and the corresponding transition state are unsuccessful so
far. It looks like that the rate-limiting factor of reduction is thetransfer of an electron to the Pt(IV) atom, not breaking of thePt−ligand bond as a result of the one-electron transfer. In thiscontext the kinetics of reduction of platinum(IV) complexes aredependent not only on the compound itself but also on the bio-reducing agent (ascorbate, glutathione, cysteine,methionine, etc.) andthe surrounding pH.27−29How these factors influence the behavior ofPt(IV) complexes will be a matter of further investigation.
QSAR Analysis. Initial Screening of the Descriptors.From the initial screening of the correlation between propertiesand biological response, it was found that the most significantdescriptors for the biological activity (as single parameters) inboth cell lines are the number of H-bond acceptors (Hacc), chargeat the Pt atom (q(Pt)), vertical and adiabatic electron affinities(Eeas and Eeas′), followed by the number of H-bond donors(Hdon) (see Table 4).
Table 2. Bond Length Changes in Complexes 24 and 28 afterthe Acceptance of an Electron in Aqueous Medium
complex
Δ bond length (Å) 24 28
Pt−O1ax 0.04 0.35Pt−O2ax 0.03 0.37Pt−N1 0.34 0.01Pt−N2 0.01 0.01Pt−Cl1/Pt−O3eq 0.04 0.04Pt−Cl2/Pt−O4eq 0.34 0.04
Figure 7. Scheme of possible reduction reactions to pentacoordinated Pt complex for M1 (bottom) and M2 (top).
Table 3. Energies (in kJ/mol) Required for Dissociation of aLigand from Complexes M1 and M2
reaction of reduction M2-ac M1-ac M1-Cl
PtIVL6 → (PtIVL5)+ + L− 305.6 303.7 246.7
PtIVL6 → (PtIIIL5)• + L• 251.3 251.6 256.9
(PtIVL6)•− = (PtIIIL6)
•− → (PtIIIL5)• + L− 124.8 113.5 88.0
Table 4. Significance of the Descriptors (as SingleParameters), Based on the Properties−Biological ResponseCorrelationa
correlation withthe response CH1 cells SW480 cells
strong (R > |0.5|) Hacc, q(Pt), Eeas′, Hdon, Eeas Hacc, q(Pt), Eeasmiddle (R < |0.5|) COOH, Es′, Es, Eea Hdon, Eeas′weak (R < |0.3|) EHOMO,MW,Ei,H/Lgap,Eis Es′,Es,COOH,MW,EHOMO,-
Eea, ELUMO, Eis, Vm, Ei
very weak(R < |0.15|)
Vm, α, μ, SASA, ELUMO α, μ, SASA, H/Lgap
afor the abbreviations see the experimental section.
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A strong correlation between MW, Vm, and α can be found.The charge at Pt, the vertical redox potential (Eeas), and thenumber of H-bond acceptors also have a strong correlation witheach other. Expectedly, there is an excellent correlation betweenEHOMO and the ionization energy (Ei) as well as among thevertical and adiabatic solvation energies. A good agreementbetween ELUMO and Eea, as well as among the HOMO/LUMOgap and first ionization energy in vacuum could be also observedduring the descriptor analyses. From a group of descriptorshaving strong correlations with each other, only a single one isexpected to contribute to a good QSAR model. With the aimof using the final QSAR model for screening purposes, it is ad-vantageous to select the descriptor in each group as one that requiresthe least computational effort, e.g., the molecular weight is mucheasier to calculate than the molecular polarizability; vertical solvationenergy can be calculated more quickly than adiabatic.The most promising models based on a single descriptor or a
combination of two, three, four, or five descriptors were chosenwith the help of simulated annealing. It was demonstrated thatthe best merit for both cell line models could be derived by com-bination of four descriptors; utilizing more than five descriptorsdecreased the merit. How R2 and Q2 of the models change withincreasing the number of the descriptors is shown in Figure S3(Supporting Information).QSARModels for the Cell Line CH1. Statistical data for the
best regression models for cytotoxicity in the cell line CH1 aresummarized in Table 5. Additional statistical information for
these and other models based on one, two, three, four, or fivedescriptors is presented in Table S4 (Supporting Information).Models derived by using only one descriptor (the good
autocorrelating q(Pt) or Hacc), expectedly have low explanatoryand predictive properties (R2 and Q2 under 50%). Essentialimprovement could be achieved by adding Hdon; R
2 increased tonearly 70%, and the predictability was over 65%, respectively.Further enhancement could be obtained by adding a thirddescriptor to the best two-variable models (Hdon and Hacc orq(Pt) and Hdon). R
2 over 75% is achieved by including theadiabatic redox potential in water (Eeas′). The developed modelsalso showed high predictive strength (Q2 > 75%) and robustness,which was proved in the external validation under severe condi-tions (Table S5, Supporting Information). Interestingly, by inclusionof the presence/absence of COOH in the combination of theHdon and Hacc model, only constitutional (easy to calculate)molecular descriptors with R2 = 75% and Q2 = 72% could beobtained (Table S4). The latter also showed good Pred.R2 on theexternal validation and can be a good alternative for screening
when quantum mechanical calculations are not possible or tooexpensive. Combining α or the autocorrelated MW with q(Pt)and Hdon gave models with moderate explanatory (∼72%) andpredictive (∼69%) properties, which totally failed in the externalvalidation, partition e where all nedaplatin derivatives (48−50)are in the predictive set (Tables S5 and S6).Reliable (R2 ≥ 80%) and predictive (Q2 ≥ 70%) models could
be constructed only by adding a fourth descriptor. The bestresults are obtained via combining polarizability with the besttwo three-descriptor models, where R2 of 86%, Q2 of 82%, andAAR < 0.4 could be achieved. Using MW (easy to calculate andhaving strong correlation with α) instead does not decrease thequality of the models. The external validation proved the robust-ness and the predictive properties and showed that the mostreliable model is built by using MW, Eeas′, Hdon, and Hacc asvariables. Developing a model by adding a fifth descriptor canslightly increase the R2 and Q2 values only when autocorrelatingdescriptors (e.g., α and MW, Eeas and Eeas′) are included, whichresults in overfitting and fake higher predictability.The complete regression equation for the final predictive
model we have chosen is as follows:
= − ′ −
− +
pIC (CH1) 0.006 MW 3.920E s 0.417H
0.363H 16.18650 ea don
acc
The plot of experimental and predicted pIC50 values of the modelis shown in Figure 8.In the PCA method all variables are combined into new
descriptors that are ranked according their ability to describe thevariation in the descriptor data. For the present case, 85% of thevariance could be explained by five components, but a QSARmodel using five components performs no better than the four-component MLR model (R2 = 0.83). Since the PCA approachrequires the calculation of all descriptors, the MLR approach isbetter suited for screening purposes. When PCA was applied onthe four descriptors (MW, Eeas′, Hdon, and Hacc) used for devel-oping our MLR model, three components, together explaining88% of the variance, were obtained (their loading plots are shownin Figure S4, Supporting Information). The score plot, obtainedby combination of the first two of them (PC1 and PC2, ex-plaining 73% of the variability), grouped well the complexes infive clusters (Figure 9). The compounds from subsets 1 and 2were split, depending on the terminal moieties of the axialligands, separating the more active esters in one cluster from theless active amides and free carboxylic acids in another. Com-pounds 3, 11, and 16, featuring terminal CH2OH and equippedwith the lowest cytotoxicity in subset 1, formed another cluster.Interestingly, nedaplatin derivatives (48−50) from subset 2grouped together with the amides and free carboxylic acids fromsubset 1, and esters 17 and 18 (having three CH2 groups spacerbetween the carbonyls in the axial chains) from subset 1 groupedtogether with amides and carboxylic acids from subset 2.
QSAR Models for the Cell Line SW480. Statistical data forthe best regression models for cytotoxicity in the SW480 cell lineare summarized in Table 6. Additional statistical information forthese and other one- to five-variable regressions is presented inTable S6 (Supporting Information).Using only one descriptor cannot give a model with good
explanatory and predictive properties (R2 and Q2 under 65%).Including a second (q(Pt) and Hdon) increases R
2 up to 73% andQ2 to 70%. However, in order to reach values over 75%, modelswith a combination of three or four descriptors should be used.Increasing the number of variables to more than four gives models
Table 5. Statistical Data for the Best Regression Models forCytotoxicity in the Cell Line CH1 of the 53 InvestigatedPt(IV) Complexes
no. ofvariables descriptors R2 Q2(LTOP) rms
1 Hacc 0.51 0.48 0.782 Hdon, Hacc 0.70 0.67 0.623 q(Pt), Eeas′, Hdon 0.79 0.75 0.534 α, q(Pt), Eeas′, Hdon 0.86 0.82 0.454 MW, q(Pt), Eeas′, Hdon 0.85 0.81 0.474 MW, Eeas′, Hdon, Hacc 0.85 0.82 0.464 α, Eeas′, Hdon, Hacc 0.85 0.82 0.465 MW, α, q(Pt), Eeas’, Hdon 0.87 0.84 0.436 MW, α, q(Pt), Eeas′, Es, Hdon 0.88 0.84 0.43
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with marginally higher R2, but this is mainly due to overfitting,since they contain autocorrelated descriptors (EHOMO or H/Lgapand Ei). The actual predictability of the best models, featuringthree, four, or five descriptors, using external validation, is sum-marized in Table S7 (Supporting Information). The lowestpredictive capability of the models could be observed on trainingsets c and e where most of the ethylenediamine derivatives or theoxaliplatin, nedaplatin, and part of the carboplatin analogues aremoved to the predictive set.The complete regression equations for the final predictive
models we have chosen are the following:
= − − −
− +
EpIC (SW480) 0.024 0.353H 0.534H
0.526 COOH 2.09050 s don acc
(1)
= − + − ′
− +
E EpIC (SW480) 0.637 4.162E s 4.522 s
0.384H 12.06050 i ea ea
don (2)
= − − +
− −
E EpIC (SW480) 1.094 2.634 4.971E s
0.404H 1.28150 i ea ea
don (3)
The plot of the predicted vs experimental pIC50 values for themodels is shown in Figure 10. Model 3 gives the best linear fit(R2 = 0.82 vs R2 =0.80 for models 1 and 2) and can predict betterthe activity of the oxaliplatin analogues (51−53), the only com-pounds from subset 2, showing some activity in the cell lineSW480 (due to the DACH carrier ligand). On the other hand,model 1 showed higher predictive R2 (near 60%) in the severecross-validation partitioning e and in addition is built from easyto calculate molecular descriptors. It is therefore favorable forscreening of new compounds.As most of the compounds from subset 2 did not show
cytotoxic activity in the SW480 cell line and IC50 could not bedetected up to 500 μM, in the current study IC50 of 600, 1000,and 2000 μMwere used as input for their cytotoxic activity. By anincrease of these values from 600 to 2000, slightly better modelswith increased R2 andQ2 could be observed (Table S8); however,the AAR values increased too and the results from the externalvalidation deteriorate slightly. The data, presented in Table 6, arebased on the optimal input IC50 of 1000 μM. The tables inSupporting Information are based on the study using IC50 = 600 μMfor complexes inactive in SW480 cells.
Figure 8. Predicted (with the selected four-variable model) vs experimental cytotoxicity in the cell line CH1. The coloring is based on the subtypescontaining the same equatorial ligands.
Figure 9. Scoring plot derived from PCA on the four descriptors (MW,Eeas′, Hdon, and Hacc) used in the proposed model for cytotoxicity in theCH1 cells: cluster I, esters from subset 1; cluster II, esters from subset 2;cluster III, amides and free carboxylic acids from subset I and nedaplatinderivatives (48−50); cluster IV, amides and free carboxylic acids fromsubset 2 and complexes 17 and 18 from subset 1; cluster V, compoundswith terminal CH2OH groups in the axial ligands.
Table 6. Statistical Data for the Best Regression Models forCytotoxicity in the SW480 Cell Line of the 53 InvestigatedPt(IV) Complexes
no. ofvariables descriptors R2 Q2(LTOP) rms
1 Hacc 0.63 0.60 0.752 q(Pt), Hdon 0.73 0.70 0.653 Es, Hdon, Hacc 0.77 0.73 0.614 Es, Hdon, Hacc, COOH 0.80 0.75 0.594 Ei, Eeas, Eeas′, Hdon 0.80 0.76 0.584 Ei, Eea, Eeas, Hdon 0.82 0.79 0.545 EHOMO, Ei, Eea, Eeas, Hdon 0.84 0.80 0.525 q(Pt), H/Lgap, Ei, Eeas, Hdon 0.82 0.78 0.566 EHOMO, Ei, Eea, Es, Eeas, Hdon 0.85 0.81 0.517 EHOMO, Ei, Eea, Es, Es′, Eeas, Hdon 0.86 0.80 0.52
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Model 2 showed the smallest difference between the predictedpIC50 values for the inactive carboplatin analogues (−2.8 ± 0.4)for input value pIC50 = −3.0 (IC50 = 1000 μM).Applying PCA, using the descriptors from the chosen four-
variable models, showed that 88−89% of the variance in the setcan be explained by three components. By plotting of the scoresof PC1 and PC3 (covering 60% of the variance) produced frommodel 3 descriptors combination, a nice clustering of the seriescould be observed (Figure 11). Similar to the clustering obtainedwith the CH1 cells model PCA, the compounds split into subsets1 and 2 esters and subsets 1 and 2 amide and free carboxylic acids.Compounds 3, 11, and 16 are again in a separate cluster, and
complexes 17 and 18 from subset 1 are in the subset 2 amides andacids cluster. In addition the EtNH2 ester derivatives (22−25)built a subcluster.
Conformational Differences. Four different conformersfor compound 2 and two different conformers for compound 50were generated (Figure S5, Supporting Information), and theirmolecular properties were calculated. The descriptors with thesmallest differences were q(Pt), α, EHOMO, H/Lgap, Ei, Eis,Eeas, and Eeas′, with RSD < 2%. A moderate effect of the con-formation was observed on Vm and SASA (RSD < 8%). Thedipole moment (μ) and solvation energies (Es and Es′) are moredependent on the conformation, where RSD rises to 17% in the
Figure 10. Predicted (with the selected four-variables model) vs experimental cytotoxicity in the SW480 cell line: top, model 1; middle, model 2;bottom, model 3. The coloring is based on the subtypes containing the same equatorial ligands.
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case of μ. The autocorrelating ELUMO and Eea showed greatdependency on the conformation, which excludes them fromthe list of descriptors able to produce reliable models. The in-fluence of the conformations of complex 2 on the predictedcytotoxicity from the best chosen four-descriptor models issummarized in Table 7.In comparison to model 1, models 2 and 3 gave better results
with respect to cytotoxicity in SW480 cells. However, the de-pendency on conformers was higher (high RSD values). Thiscircumstance is expected, since the conformation has a significantimpact on descriptor Eea. The vertical and adiabatic redox potentialsinwater have small conformational dependence but also close valuesin the series, which pronounce the effect of the conformation to thepredicted cytotoxicity.Free−Wilson QSAR Model. In order to judge the
contribution of different substituents to the biological activityof the compounds, Free−Wilson QSAR models for cytotoxi-city of the complexes in the CH1 and SW480 cell lines weredeveloped. The models are based on the concept that eachsubstituent makes an additive and constant contribution to thebiological activity regardless of substituent variation in the rest ofthe molecule.30 Each compound was presented as a binary stringwith a length of 24 substituents (the different equatorial ligands,spacers between the two carbonyls, and terminal functionalgroups in the axial ligands). A term is equal to 1when a substituent ispresent at a particular position and 0 when it is absent. Thecontribution of each substituent was calculated using MLR, and the
following models were obtained:
= − +
+ −+ −+ −− −+ +− ++ ++ +−−++−−
i
pIC (CH1) 0.135A(NH ) 0.025A(en)
0.212A(EtNH ) 0.049A(DACH)
0.259L(Cl) 0.280L(CBDA)
0.082L(glyc) 0.049L(ox)
0.010X(succ) 0.104X(Glu)
0.107X(MeGlu) 0.053X(DiMeGlu)
0.277R(COOH) 0.053R(COOMe)
0.167R(COOEt) 0.185R(COOPr)
0.184R(COO Pr) 0.209R(COOBut)
0.104R(CONHProp)
0.014R(CONHcp)
0.022R(CONHch)
0.016R(CONHBz)
0.232R(CONH(CH ) OH)
0.021R(CONH(CH ) OMe)
50 3
2
2 2
2 2
for which R2 = 0.90, Q2 = 0.76, and AAR = 0.28.
= − −
+ ++ −− +− −+ +− −+ +++−−++−+
i
pIC (SW480) 0.140A(NH ) 0.034A(en)
0.223A(EtNH ) 0.061A(DACH)
0.341L(Cl) 0.351L(CBDA)
0.056L(glyc) 0.061L(ox)
0.053X(succ) 0.038X(Glu)
0.094X(MeGlu) 0.048X(DiMeGlu)
0.195R(COOH) 0.007R(COOMe)
0.146R(COOEt) 0.177R(COOPr)
0.085R(COO Pr)
0.206R(COOBut)
0.066R(CONHProp)
0.036R(CONHcp)
0.012R(CONHch)
0.012R(CONHBz)
0.199R(CONH(CH ) OH)
0.012R(CONH(CH ) OMe)
50 3
2
2 2
2 2
Figure 11. Score plot derived from PCA using four descriptors (Ei, Eea,Eeas, and Hdon), applied for modeling the cytotoxicity in SW480 cells(model 3): cluster I, compounds with a terminal free CH2OH group inthe axial ligands; cluster II, amides and free carboxylic acids from subset1; cluster III, amides and free carboxylic acids from subset 2 and 17 and18 from subset 1; cluster IV, esters from subset 1 (without the EtNH2derivatives); cluster V, esters from subset 1/the EtNH2 derivatives;cluster VI, esters from subset 2.
Table 7. Average IC50 Values for Conformers of Complex 2, Derived from the Best Four-Variable QSAR Models, for CH1 andSW480 Cells in Comparison to Experimental Dataa
cell line CH1 RSD, % SW480 model 1 RSD, % SW480 model 2 RSD, % SW480 model 3 RSD, %
exptl 0.62 ± 0.32 52 3.8 ± 1.0 26 3.8 ± 1.0 26 3.8 ± 1.0 26linear fit 0.59 ± 0.46 80 10.9 ± 4.9 45 8.3 ± 8.6 104 4.6 ± 4.8 104cross-validat predictions (LOOP) 0.60 ± 0.50 83 12.6 ± 6.4 52 8.6 ± 9.3 108 5.0 ± 5.5 110ext validationb 0.91 ± 0.77 85 35.2 ± 19.5 55 42.3 ± 10.6 25 3.7 ± 3.6 97
aResults are presented as the mean ± sd. bValues from partitioning, where all the conformers are in the training set.
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for which R2 = 0.91, Q2 = 0.80, AAR = 0.26.In the above equations, A is the carrier ligand, L the leaving
groups, X the spacer between the two carbonyl groups in the axialligands, and R the terminal functional group of the axial chains.Explanation of results and predictability of the models with thegiven set of substituents is good. The highest positive effecton the cytotoxicity in both cell lines have A = EtNH2, L = Cl,R= COOEt, COOPr and COOBut. The lowest cytotoxic effectis due to A = NH3, L = CBDA, R = COOH, and CONH-(CH2)2OH. The spacers between the carbonyl groups in the axialchains have a lower impact on the cytotoxicity in the series. In themodel for SW480 cells, A = DACH and L = ox have a slightlypositive effect on the cytotoxicity, contrary to the model for CH1cells. In contrast, R = COOiPr has a much higher positive effecton the pIC50 values in the cell line CH1 than in SW480 cells.
■ CONCLUSIONSReliable, robust, and predictive four-variable models for the invitro cytotoxicity of bis-, tris-, and tetrakis(carboxylato)platinum-(IV) complexes in cisplatin sensitive CH1 cells and intrinsicallycisplatin resistant SW480 cells were developed. The QSARmodel of choice (R2 = 85%, Q2 = 82%) for CH1 cells was builtusing the combination of MW, Hdon, Hacc and Eeas′. For theSW480 cell line, models consisting of Es, Hdon, Hacc, and COOH(R2 = 80%, Q2 = 75%) and Ei, Eeas, Eeas′, and Hdon (R
2 = 80%,Q2 = 76%) were proposed. The autocorrelating descriptorsq(Pt), Hacc, and Eeas distinguished well the two main subtypes ofcompounds, namely, bis(carboxylato)dichlorido (subset 1) fromtris- and tetrakis(carboxylato) (subset 2) complexes and showedsome minor discrimination within the subsets, depending on dif-ferent equatorial ligands. Hacc predicted a slightly higher activityfor nedaplatin analogues compared to the other compounds insubset 2 (the case of CH1 cells), while q(Pt) and Eeas dis-criminated oxaliplatin analogues as more active (the case ofSW480 cells). The constitutional descriptor Hdon discriminatedthe main functionalities on the axial ligands: amides and freecarboxylic acids from esters. Therefore, the latter is crucial forbuilding a good model. MW as a descriptor indicates the increaseof lipophilicity (respectively, cytotoxicity) in the series withincreasing the size of the axial chains or the size of equatorialamines. Eeas′ and Es, redox behavior and solubility correspond toimportant physicochemical parameters of Pt(IV) complexes andexpectedly show significance for the prediction of the biologicalresponse. The results of the study represent a step toward a betterunderstanding of the biological behavior of Pt(IV) carboxylatocomplexes and their further rational development.
■ EXPERIMENTAL SECTIONAll reagents and solvents were obtained from commercial suppliers andwere used without further purification. Water was purified throughreverse osmosis, followed by double distillation. For column chro-matography, silica gel 60 (Fluka) was used. 1H, 13C, 15N, 195Pt, and two-dimensional 1H−1H COSY, 1H−13C and 1H−15N HSQC, and 1H−13CHMBCNMR spectra were recorded with a Bruker Avance III 500 MHzNMR spectrometer at 500.32 MHz (1H), 125.81 MHz (13C), 107.55MHz (195Pt), and 50.70 MHz (15N) in DMF-d7 or D2O (in the case ofnedaplatin and its dihydroxido Pt(IV) analogue) at ambient temper-ature. The splitting of proton resonances in the 1H NMR spectra aredefined as follows: s = singlet, bs = broad singlet, d = doublet, t = triplet,and m = multiplet. 15N chemical shifts were referenced relative toexternal NH4Cl, whereas
195Pt chemical shifts were referenced relativeto external K2[PtCl4] (see Figure 2, compounds 48−50, includingNMRnumbering scheme). IR spectra were recorded with a Bruker Vertex70 FT-IR spectrometer (4000−400 cm−1) by using an ATR unit.
Intensities of reported IR bands are defined as follows: br = broad, s =strong, m = medium, and w = weak. Electrospray ionization massspectrometry was carried out with a Bruker Esquire 3000 instrumentusing MeOH/H2O as solvent. Elemental analyses were performed usinga Perkin-Elmer 2400 CHN elemental analyzer at the MicroanalyticalLaboratory of the University of Vienna, Austria, and are within±0.4% ofthe calculated values, confirming their ≥95% purity (see Table S9,Supporting Information).
Synthesis and characterization of complexes 1−47 and 51−53 aredescribed in refs 14, 17, and 20. The synthetic procedure for compounds48−50, their precursor nedaplatin, and its dihydroxidoplatinum(IV)analogue is reported herein. Synthesis and characterization of com-pound 48 was reported recently.22
(SP-4-3)-Diammineglycolatoplatinum(II) (Nedaplatin). Neda-platin was prepared starting from K2PtCl4 via cis-Pt(NH3)2I2. Anamount of 1.206 g (2.4972 mmol) of the latter was suspended in 36 mLof triply distilled water, and 870 mg (4.7564 mmol) of silver glycolatewere added. The suspension was left stirring overnight in the dark, andthen the obtained silver iodide was filtered through a sintered glassfunnel with a filter paper disk (MNGF-3). The clear solution was stirredat room temperature in the dark for 4 h, and then Amberlite·HCl(conditioned with NaOH to its OH form) was added slowly in smallportions to the solution of the complex while stirring until pH (9−10)was achieved. The mixture was left stirring overnight in the dark. ThenAmberlite and traces of reduced Pt(0) were filtered off through asintered glass funnel with a filter paper disk (MN GF-3). The volume ofthe filtrate was reduced and cooled in the fridge. The obtained pre-cipitate was filtered off, washed with acetone, and dried in a vacuumdesiccator over P2O5 to yield 498 mg of a white to pale yellow solid. Yield:498 mg (69%). 1H NMR: δ = 4.02 (s (with Pt satellites) H-1) ppm. 13CNMR: δ = 194.8 (C-2), 68.1 (C-1) ppm. 195Pt NMR: δ = −47 ppm. IR(ATR): ν= 3201 br, 2995 br (νN−H); 2889 br; 1613 s, 1578 s (νCO); 1443w; 1337 s, 1319 m, 1060 w cm−1. Anal. (C2H8N2O3Pt) C, H, N.
(OC-6-44)-Diammineglycolatodihydroxidoplatinum(IV). Ne-daplatin (1.0995 g, 3.6267 mmol) was suspended in 22 mL of triplydistilled water, and then an amount of 11 mL of 30% H2O2 was added.The mixture was stirred for 3 h at 30 °C (in the dark). The volume of theclear yellow solution obtained was reduced on a rotavapor, cooled in thefridge, and then precipitated with a sufficient amount of cold acetone.The precipitation was finalized with the help of ultrasonic waves andthen the final product was filtered off, washed with acetone, and dried invacuo to obtain a white to pale yellow solid. Yield: 1.3150 g (94%). 1HNMR: δ = 4.30 (s + d, 3JPt,H = 20.8 Hz, H-1) ppm.
195Pt NMR: δ = 3222ppm. IR (ATR): ν = 3462 br (νPtO‑H); 3229 br, 3045 br (νN−H); 2791 w;1653 s, 1588 m (νCO); 1346 s, 1308 s; 1060 w cm−1.
(OC-6 -42 ) -D iammineb i s (3 - ca rboxypropanoato ) -glycolatoplatinum(IV) (48). Succinic anhydride (950mg, 9.4934mmol)and 800 mg (2.0607 mmol) of (OC-6-44)-diammineglycolatodihydro-xidoplatinum(IV) were suspended in dry DMF (26 mL), and thereaction mixture was stirred at 60 °C for 8 h. During this time, the solidmaterial dissolved to form a pale yellow solution. DMF was thenremoved under reduced pressure. The residue was suspended in acetonewith the help of ultrasonic waves, filtered off, and washed with acetone.The pale yellow solid obtained was then dried in vacuo. Yield: 1.0865 g(98%). 1H NMR: δ = 12.36 (bs, 2H, COOH), 6.47 (m, 3H, NH3), 6.05(bs, 3H, NH3), 4.08 (bs, 2H, H-1), 2.56 (t,
3JH,H = 6.5 Hz, 4H, H-4 orH-5), 2.49 (t, 3JH,H = 6.5 Hz, 4H, H-4 or H-5) ppm.
13C NMR: δ = 187.0(C-2), 180.1 (C-3 or C-6), 174.0 (C-3 or C-6), 70.8 (C-1), 30.6 (C-4 orC-5), 29.8 (C-4 or C-5) ppm. 15N NMR: δ = −58.6, −50.7 ppm. 195PtNMR: δ = 3460 ppm. IR (ATR): ν = 3201 br, 3109 br (νN−H); 2934 br;1715 m, 1654 s, 1620 s, 1574 s (νCO); 1405 w; 1340 s, 1307 s, 1242 m,1198 m, 1162 m, 1049 w cm−1. Anal. (C10H18N2O11Pt) C, H, N.
(OC-6-42)-Diammineglycolatobis((4-propyloxy)-4-oxobutanoato)platinum(IV) (49). CDI (253 mg, 1.5566 mmol) indry DMF (9 mL) was added to a solution of 48 (408 mg, 0.7593 mmol)in dry DMF (10 mL), and the mixture was heated to 60 °C. After 10 minof being stirred, the solution was cooled to room temperature and CO2was removed by flushing with argon. Sodium propanolate in n-propanol(9 mL) (a piece of Na (15 mg), dissolved in 10 mL of n-propanol) wasadded to the solution and stirred for 30 h at room temperature. Then
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propanol and DMF were removed under reduced pressure to form ayellow oil. The crude product was purified by column chromatography(EtOAc/MeOH, 4:1), then isolated from an EtOAc suspension, anddried in vacuo to yield a white to pale yellow powder. Yield: 95 mg(20%). 1H NMR: δ = 6.57 (m, 3H, NH3), 6.12 (bs, 3H, NH3), 4.06 (bs,2H, H-1), 4.01 (t, 3JH,H = 6.5 Hz, 4H, H-7), 2.57 (m, 4H, H-4), 2.53 (m,4H, H-5), 1.62 (m, 4H, H-8), 0.91 (t, 3JH,H = 7.2 Hz, 6H, H-9) ppm.
13CNMR: δ = 186.9 (C-2), 179.8 (C-3), 172.6 (C-6), 70.8 (C-1), 65.6(C-7), 30.5 (3JPt,C = 41.0 Hz, C-4), 29.9 (C-5), 21.9 (C-8), 9.9 (C-9)ppm. 15N NMR: δ = −58.3, −49.7 ppm. 195Pt NMR: δ = 3464 ppm. IR(ATR): ν = 3301 br, 3212 br, 3048 br (νN−H); 2971 br; 1729 m, 1707 s,1621 s, 1585 m (νCO); 1483 w; 1435 m, 1345 m, 1316 s, 1249 w,1180 s, 1166 m, 1105 m, 984 w cm−1. ESI MS (positive): m/z 643.9[M +Na+]+. ESIMS (negative):m/z 620.8 [M−H+]−, 656.7 [M +Cl−]−.Anal. (C16H30N2O11Pt·0.5H2O) C, H, N.(OC-6-42) -D iamminebis ( (4 -cyc lopenty lamino) -4 -
oxobutanoato)glycolatoplatinum(IV) (50). CDI (176 mg, 1.0835mmol) in dry DMF (7 mL) was added to a solution of 48 (284 mg,0.5285mmol) in dry DMF (9mL), and the mixture was heated to 60 °C.After 10 min of being stirred, the solution was cooled to room tem-perature and CO2 was removed by flushing with argon. Cyclopentyl-amine (115 μL, 1.1628 mmol) in 4 mL of dry DMF was added to thesolution and stirred for 24 h at room temperature (the solution changedto a yellow suspension). DMF was removed under reduced pressure toform a pale brown solid. The crude product was purified by columnchromatography, using EtOAc/MeOH = 2:1, and subsequently isolatedfrom an EtOAc suspension, washed with EtOAc and Et2O, and dried invacuo to yield an almost white solid. Yield: 116 mg (33%). 1HNMR: δ =7.78 (d, 3JH,H = 6.6 Hz, 2H, CONH), 6.49 (m, 3H, NH3), 6.06 (bs, 3H,NH3), 4.09 (m, 2H, H-7), 4.06 (s, 2H, H-1), 2.50 (t,
3JH,H = 7.4 Hz, 4H,H-4 or H-5), 2.36 (t, 3JH,H = 7.3 Hz, 4H, H-4 or H-5), 1.83 (m, 4H, H-8),1.66 (m, 4H, H-9), 1.52 (m, 4H, H-9), 1.44 (m, 4H, H-8) ppm. 13CNMR: δ = 187.0 (C-2), 180.7 (C-3), 171.2 (C-6), 70.9 (C-1), 50.8(C-7), 32.5 (C-8), 31.8 (C-4 or C-5), 31.5 (C-4 or C-5), 23.6 (C-9) ppm.15N NMR: δ = −58.7, −50.2, 106.7 ppm. 195Pt NMR: δ = 3459 ppm. IR(ATR): ν = 3269 br, 3058 br (νN−H); 2958 br; 1697 m, 1632 m, 1571 s(νCO); 1483 m; 1441 s, 1339 w, 1319 m, 1255 m, 1195 m, 1106 s, 997w cm−1. ESI MS (positive): m/z 694.0 [M + Na+]+, 671.0 [M + H+]+.ESI MS (negative): m/z 669.9 [M − H+]−, 705.8 [M + Cl−]−. Anal.(C20H36N4O9Pt·0.5H2O) C, H, N.Crystallographic Structure Determination. Yellow crystals of 7,
suitable for X-ray data collection, were obtained after slow evaporationof a MeOH/EtOAc solution. X-ray diffraction measurement was per-formed on a Bruker X8 APEXII CCDdiffractometer. A single crystal waspositioned at 40mm from the detector, and 1638 frames were measured,each for 15 s over 1° scan width. The data were processed using SAINTsoftware.31 Crystal data, data collection parameters, and structurerefinement details are given in Table S10 (Supporting Information).The structure was solved by direct methods and refined by full-matrixleast-squares techniques. Non-H atoms were refined with anisotropicdisplacement parameters. H atoms were inserted in calculated positionsand refined with a riding model. The isotropic thermal parameters wereestimated to be 1.2 times the values of the equivalent isotropic thermalparameters of the atoms to which hydrogens were bonded. Structuresolution was achieved with SHELXS-97 and refinement with SHELXL-97,32 and graphics were produced with ORTEP-3.33
CytotoxicityAssays.CH1 (ovarian carcinoma, human) cells were agift from Lloyd R. Kelland (CRC Centre for Cancer Therapeutics,Institute of Cancer Research, Sutton, U.K.). SW480 (colon carcinoma,human) cells were kindly provided by Brigitte Marian (Institute ofCancer Research, Department of Medicine I, Medical University ofVienna, Austria). Cells were grown in 75 cm2 culture flasks (Iwaki/AsahiTechnoglass) as adherent monolayer cultures in complete medium,i.e., minimal essential medium (MEM) supplemented with 10% heat-inactivated fetal bovine serum, 1 mM sodium pyruvate, 4 mML-glutamine, and 1% v/v nonessential amino acids (from 100× ready-to-use stock) (all purchased from Sigma-Aldrich) without antibiotics.Cultures were maintained at 37 °C in a humidified atmospherecontaining 5% CO2 and 95% air. Cytotoxicity in the cell linesmentioned above was determined by the colorimetric MTT assay
(MTT = 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bro-mide, purchased from Fluka). Cells were harvested from culture flasksby trypsinization and seeded in 100 μL aliquots in complete mediuminto 96-well microculture plates (Iwaki/Asahi Technoglass) in thefollowing densities to ensure exponential growth of untreated controlsthroughout the experiment: 1.5 × 103 (CH1) and 2.5 × 103 (SW480)viable cells per well. Cells were allowed to settle and resume exponentialgrowth in drug-free complete culture medium for 24 h, followed by theaddition of dilutions of the test compounds in 100 μL/well of the samemedium. After continuous exposure for 96 h, the medium was replacedby a 100 μL/well RPMI 1640 medium (supplemented with 10% heat-inactivated fetal bovine serum and 4 mM L-glutamine) plus 20 μL/wellsolution of MTT in phosphate-buffered saline (5 mg/mL) (all purchasedfrom Sigma-Aldrich). After incubation for 4 h, medium/MTT mixtureswere removed, and the formazan product formed by viable cells wasdissolved in DMSO (150 μL/well). Optical densities at 550 nm were mea-sured with a microplate reader (Tecan Spectra Classic), using a referencewavelength of 690 nm to correct for unspecific absorption. The quantity ofviable cells was expressed as the percentage of untreated controls, and 50%inhibitory concentrations (IC50) were calculated from concentration−effectcurves by interpolation. Evaluation is based on the mean from three in-dependent experiments, each comprising triplicates per concentration level.
Theoretical Calculations. All calculations were performed with theGaussian 09 software package.34 The starting structures foroptimizations (complexes 1, 6, 7, 22, 38) were taken from the availableX-ray data;14,17,18,20 the crystal structure of complex 7 is reported herein.The other compounds were modeled by modification of the latter. Acomplete conformational search is not feasible for systems with as manydegrees of freedom as the present. Furthermore, there are very few forcefields capable of handling Pt complexes. In order to see the influence ofdifferent conformations to the calculated parameters and to the QSARmodels, four different conformers of compound 2 and two of compound50 were modeled and their geometry was optimized (Figure S5,Supporting Information). In principle, the possible conformationaluncertainties are in the chains of the axial ligands because the surround-ings around the platinum atoms were taken from the crystallographicdata. For compounds 19, 20, 42, 43, and 44, which have two chiralcenters in the axial carbon chains, the meso RS forms were taken for thecalculations. These compounds were tested for cytotoxicity as a mixtureof RR/SS/RS = 1:1:2 stereoisomers. In the particular case, stereo-chemistry will not affect essentially the activity because the chiral centersare in the middle of the carbon chains of the axial ligands, which aresupposed to be lost after the activation of the complexes via reduction invivo. In contrast, in the case of oxaliplatin, featuring DACH (1,2-diaminocyclohexane), the R,R configuration of the ligand should berespected (compounds 51−53).
The DFT long-range corrected hybrid wb97x functional was used forall calculations35 in connection with the Def2-SVP basis set36 witheffective core potential37 for optimizing the geometries and calculationof themolecular descriptors. For the calculations of polarizability and dipolemoment the basis set were augmented by a set of diffuse functions.
Geometry optimizations were performed in the gas phase and in awater solvent model by using the IEFPCM38 method. Solvent accessiblesurface area (SASA) was extracted after single point energy calculationof the gas optimized structures in water environment with the ipcm39
method, where the cavity is defined by a self-consistent isodensitycontour in a water solvent model. Atomic charges were calculated usingthe NPA approach.40
The molar volume and the HOMO and LUMO energies were takenfrom the gas phase optimized geometries. The energies of solvation werecalculated by extracting the energies in water environment (using theiefpcm method) with (adiabatic Es′) or without (vertical Es) optimizationfrom the total energies in gas phase. For estimation of the ionizationpotential and electron affinity, the energies of the corresponding anion andcation radicals were calculated in the gas phase and in solvent, with (only forthe anion radicals) and without geometry optimization.
QSAR Analysis. QSAR Data Set. The pIC50 = log(1/IC50) values,used to develop the QSAR models, were taken from the MTT assays,described in refs 14 and 18−20 for complexes 1−47 and 51−53 and inthe present paper for complexes 48−50. The cytototoxicity data in CH1
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and SW480 cells for all investigated compounds in comparison with theclinically approved platinum-based drugs cisplatin, carboplatin,oxaliplatin, and nedaplatin are summarized in Table 1.By use of QM calculations, the following descriptors were extracted:
molar volume (Vm), dipole moment (μ), polarizability (α), charge onthe Pt atom (q(Pt)), energy of the HOMO and LUMO (and the corre-sponding HOMO−LUMO gap), SASA, vertical and adiabatic solvationenergies (Es and Es′), vertical gas-phase ionization energies (Ei) andelectron affinities (Eea), and vertical and adiabatic oxidation (Eis) andreduction (Eeas and Eeas′) potentials in the water solvent model. Inaddition molecular weight (MW), number of H-bonds donors (Hdon),numbers of H-bonds acceptors (Hacc), and presence/absence ofcarboxylic groups (COOH) in the axial ligands as constitutional moleculardescriptors were used. The values of the used descriptors in the presentstudy are summarized in Tables S3 and S4 (Supporting Information).Chemometric Methods and Statistics. QSAR analysis was
performed with the QSAR program41 developed by the Ponder groupand Schrodinger Strike 2.0 for Maestro application.42 Standard multiplelinear regression (MLR) and principal component analysis (PCA)methods were used to analyze the data, and simulated annealing wasemployed to identify the best combinations of descriptors. All de-scriptors were centered and autoscaled prior to analysis.The robustness of the models and their predictivity were evaluated
through R2, Q2 (R2 of cross-validated predictions, using the leave-one-out procedure (LOOP) or leave-two-out procedure (LTOP)), AAR(average absolute error), and rms (root mean squared error). The actualpredictive capability of every model was checked with external validationby splitting the data set into training and predictive sets. Five differentways of partitioning the data into training and predictive data sets wereused in order to test the robustness of the QSARmodel. In each case thetraining set encompassed 75% of the data while the remaining 25% wasselected as (a) random, including 14 compounds representing everysubtype, (b) including cisplatin and its bis(ethylamine) analoguederivatives (complexes 1−5, counting all conformers for 2 and 21−26),(c) including most of the ethylenediamine analogues (complexes 6−19), (d) including most of the carboplatin analogues (27−40), and (e)including oxaliplatin, nedaplatin, and the other part of the carboplatinanalogues (complexes 41−53, counting both conformers of 50). By useof the models derived from the training sets, the pIC50 values in thepredictive sets were calculated and R2 predictive, AAS, and RMS weremeasured.
■ ASSOCIATED CONTENT*S Supporting InformationFigures with ORTEP view of 7, chemical structures of the modelsystems used in the reduction studies, dependency of the QSARmodel properties from the descriptors number, loading plotsderived from the PCA of the CH1 cells four variable model, andsuperposition of the optimized conformers of complexes 2 and50; tables with comparison of calculated and experimentalgeometrical parameters, calculated descriptors, and additionalstatistical data for chosen QSAR models and their externalvalidation, dependency of the QSAR model properties from theinput IC50 values used for the inactive compounds, elementalanalysis data, structure refinement details for 7; X-ray crystallo-graphic data in CIF format. This material is available free ofcharge via the Internet at http://pubs.acs.org. Crystallographicdata have been deposited with the Cambridge CrystallographicData Center with number CCDC 909001. Copies of data can beobtained, free of charge, on application to CCDC, 12 UnionRoad, Cambridge CB2 1EZ, U.K. ([email protected]).
■ AUTHOR INFORMATIONCorresponding Author*For F.J.: phone, +45-87155908; e-mail, [email protected]. ForM.G.: phone, +43-1-4277-52600; fax, +43-1-4277-52680; e-mail,[email protected].
NotesThe authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
H.P.V. is thankful for financial support from the University ofVienna, Austria, within the doctoral program InitiativkollegFunctional Molecules IKI041-N. The authors are indebted to theFFGResearch and Technology Development, the FWF(Austrian Science Fund, Grant P20683-N19), and COST D39.This work was supported by grants from the Danish Center forScientific Computation and theDanish Natural Science ResearchCouncil. We also thank Mahsa S. Adib-Razavi for performing theMTT tests for compounds 48−50.
■ ABBREVIATIONS USED
FDA, Food and Drug Administration; ESP, electrostaticpotential; SAR, structure−activity relationship; QSAR, quanti-tative structure−activity relationship; QSPR, quantitativestructure−property relationship; DFT, density functionaltheory; MLR, multiple linear regression; MM, molecularmechanics; log Po/w, logarithm of partition coefficient betweenn-octanol and water; Ep, redox potential; NPA, naturalpopulation analysis; PC, principal component; PCA, principalcomponent analysis; RSD, relative standard deviation; CDI, 1,1′-carbonyldiimidazole
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S1
Supporting Information
Theoretical investigations and density functional
theory based quantitative structure activity
relationships model for novel cytotoxic Pt(IV)
complexes
Hristo Varbanov†, Michael A. Jakupec†, Alexander Roller†, Frank Jensen�*, Markus Galanski†*,
and Bernhard K. Keppler†
† University of Vienna, Institute of Inorganic Chemistry, Währinger Strasse 42, A-1090 Vienna,
Austria
� University of Aarhus, Department of Chemistry, Langelandgade 140, 8000 Aarhus C, Denmark
122
S2
Contents
Page S3 Figure S1. Ortep view of 7.
Page S4 Figure S2. Chemical structure of model systems M1 and M2.
Page S4 Figure S3. Dependency of the linear fit (R2) and predictability (Q2) of the QSAR
models from the number of descriptors used for CH1 and for SW480 cells.
Page S5 Figure S4. Loading plots derived from PCA on the four descriptors, used in the
proposed model (MW, Eeas’, Hdon and Hacc).
Page S5 Figure S5. Superposition of the optimized four conformers of complex 2 and two
conformers of complex 50.
Page S6 Table S1. Comparison of crystal structure and wb97x optimized geometries in the
gas phase and in a solvent model for 1, 6, 7, 22 and 38.
Page S8 Table S2. Calculated descriptors for the investigated compounds (1).
Page S11 Table S3. Calculated descriptors for the investigated compounds (2).
Page S14 Table S4. QSAR models for the CH1 cell line with simulated annealing chosen
combination of descriptors.
Page S16 Table S5. External validation of the best models for cytotoxicity in CH1 cells,
obtained with three, four or five descriptors.
Page S18 Table S6. QSAR models for the SW480 cell line with simulated annealing chosen
combination of descriptors.
Page S20 Table S7. External validation of the best models for SW480 cells, obtained with
three, four or five descriptors.
Page S22 Table S8. Statistical parameters for the best four-variable models, using IC50 =
600, 1000 or 2000 μM as input for the inactive compounds from subset 2.
Page S22 Table S9. Elemental analysis data.
Page S23 Table S10. Crystal data and details of data collection for 7.
123
S3
Figure S1. ORTEP view of 7 with atom labeling scheme. The thermal ellipsoids have been
drawn at 50% probability level.
124
S4
Figure S2. Chemical structure of model systems M1 and M2
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
0 1 2 3 4 5 6 7
R2
Q2
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
0 1 2 3 4 5 6 7 8
R2
Q2
Figure S3. Dependency of the linear fit (R2) and predictability (Q2) of the QSAR models from
the number of descriptors used for CH1 (left) and for SW480 (right) cells.
125
S5
Figure S4. Loading plots derived from PCA on the four descriptors (MW, Eeas’, Hdon and Hacc),
used in the proposed model for cytotoxicity in CH1 cells.
Figure S5. Superposition of the optimized four conformers of complex 2 (left) and two
conformers of complex 50 (right).
�sd= ± 30 kJ mol �sd= ± 5 kJ mol
126
S6
Table S1. Comparison of crystal structure and wb97x optimized geometries in the gas phase and
in a solvent model for 1, 6, 7, 22 and 38.
bond lengths(Å), angles(º) 1 6 7 22 38
X-ray 2.050, 2.066
2.054, 2.054
2.050, 2.036
2.063, 2.068
2.050, 2.050
DFT/gas 2.074, 2.085
2.085, 2.085
2.088, 2.076
2.107, 2.100
2.073, 2.073
Pt-N
DFT/solv 2.062, 2.055
2.058, 2.058
2.060, 2.053
2.076, 2.080
2.050, 2.051
X-ray 2.311, 2.319
2.318, 2.318
2.338, 2.307
2.309, 2.324
2.005, 2.031
DFT/gas 2.310, 2.321
2.307, 2.307
2.318, 2.297
2.319, 2.306
1.965, 1.966
Pt-Cl/Pt-Oeq
DFT/solv 2.340, 2.334
2.338, 2.338
2.328, 2.348
2.335, 2.343
1.988, 1.990
X-ray 1.993, 2.008
2.011, 2.011
2.000, 2.031
2.039, 2.039
1.997, 1.965
DFT/gas 2.013, 2.018
2.016, 2.016
2.012, 2.018
1.998, 2.030
2.011, 2.017
Pt-Oax
DFT/solv 2.013, 2.016
2.015, 2.015
2.017, 2.012
2.001, 2.025
2.011, 2.017
X-ray 90.2 83.7 83.3 92.7 93.1
DFT/gas 91.0 83.1 83.5 95.9 93.2 N-Pt-N
DFT/solv 90.1 83.4 83.4 93.9 91.3
X-ray 94.4 91.2 89.6 92.2 95.1
DFT/gas 95.1 94.7 94.1 94.4 97.7 Cl-Pt-Cl/
Oeq-Pt-Oeq
DFT/solv 93.7 93.0 92.4 93.8 95.4
Oax-Pt-Oax X-ray 172.6 174.5 168.7 172.7 171.8
127
S7
DFT/gas 175.7 176.0 172.3 171.1 173.9
DFT/solv 174.6 176.8 172.1 171.8 173.1
X-ray 123.0, 125.3
125.5, 125.5
125.2, 126.7
123.7, 127.5
123.3, 127.7
DFT/gas 122.6, 122.4
126.5, 126.5
126.8, 126.0
126.3, 126.7
122.8, 122.2
Pt-Oax-C
DFT/solv 123.0, 123.3
126.2, 126.2
126.4, 125.9
128.6, 126.8
122.9, 123.3
X-ray - 108.2, 108.2
109.2, 109.5
120.2, 118.7 -
DFT/gas - 108.2, 108.3
109.0, 107.5
120.6, 118.1 -
Pt-N-C
DFT/solv - 108.8, 108.8
108.3, 109.9
118.5, 120.5 -
128
S8
Table S2. Calculated descriptors for the investigated compounds: molecular weight (MW),
molar volume (Vm), polarizability (�), solvent accessible surface area (SASA), dipole moment
(�), charge at the Pt atom (q(Pt)), vertical and adiabatic energy of hydration (Es and Es’), number
of H-bond donors and acceptors (Hdon and Hacc), presence (1) or absence (0) of COOH group.
The coloring is based on the subtypes, exerting the same equatorial ligands (Figure 2)
MW Vm � SASA � q (Pt) Es Es' complex
(g/mol) (cm3/mol) (bohr3) (bohr2) (D) (a.u.) (kJ/mol) (kJ/mol) Hdon Hacc COOH
1 534.22 222.60 215.54 1121.38 5.217 0.913 86.035 89.785 4 8 1
2a 590.32 280.04 266.29 1105.02 4.895 0.919 78.408 82.114 2 8 0
2b 590.32 318.18 270.31 1034.83 4.996 0.917 92.614 96.406 2 8 0
2c 590.32 278.93 264.05 1143.75 6.813 0.917 71.440 75.377 2 8 0
2d 590.32 308.56 265.24 1232.36 6.339 0.918 70.790 74.999 2 8 0
3 620.35 295.30 284.66 1490.84 2.696 0.922 110.316 115.194 6 10 0
4 616.41 320.11 300.92 1240.14 3.139 0.923 90.276 95.023 4 8 0
5 668.49 351.10 340.09 1270.93 4.711 0.924 92.679 97.081 4 8 0
6 560.24 262.29 235.78 1249.41 10.038 0.883 111.868 119.697 4 8 1
7 588.30 279.03 263.82 1333.65 9.631 0.885 107.458 114.152 2 8 0
8 616.35 303.87 289.57 1465.76 9.456 0.885 107.375 114.120 2 8 0
9 644.40 350.94 314.84 1583.99 9.475 0.885 107.265 113.794 2 8 0
10 672.45 344.68 340.01 1866.04 9.426 0.885 107.383 113.902 2 8 0
11 646.38 313.16 308.55 1232.00 11.048 0.885 138.603 146.752 6 10 0
12 642.43 325.44 324.77 1368.51 9.604 0.885 122.957 130.222 4 8 0
13 694.51 357.09 364.41 1451.39 9.768 0.885 122.864 130.239 4 8 0
14 588.31 271.41 262.25 1272.38 5.813 0.884 105.143 110.849 4 8 1
129
S9
15 644.42 320.71 312.35 1313.59 5.582 0.884 99.242 104.679 2 8 0
16 674.45 351.78 330.49 1229.71 4.147 0.885 129.568 135.961 6 10 0
17 670.50 340.28 339.25 1407.21 12.531 0.886 97.084 102.165 4 8 0
18 722.58 407.88 380.11 1339.52 9.977 0.886 104.990 110.228 4 8 0
19 644.40 321.27 311.74 1261.18 6.453 0.883 97.187 102.575 2 8 0
20 672.46 356.75 336.82 1203.31 6.300 0.884 97.318 102.509 2 8 0
21 590.32 277.70 269.56 1432.61 6.616 0.900 137.789 145.133 4 8 1
22 618.38 331.49 296.10 1714.83 9.048 0.899 111.916 118.178 2 8 0
23 646.43 296.25 321.51 1911.50 10.306 0.897 116.950 123.075 2 8 0
24 674.49 372.49 346.95 1425.27 10.418 0.897 116.643 123.350 2 8 0
25 674.49 371.93 345.56 1406.09 10.369 0.897 115.271 121.218 2 8 0
26 724.59 381.31 396.75 1879.12 5.916 0.899 124.879 131.664 4 8 0
27 605.42 298.95 264.44 1311.26 6.574 1.330 117.073 123.275 4 12 1
28 633.48 307.57 291.45 1320.90 9.854 1.330 109.316 115.441 2 12 0
29 661.53 338.83 316.66 1284.74 9.881 1.330 107.975 114.256 2 12 0
30 689.58 379.24 342.06 1310.12 9.949 1.330 107.649 114.011 2 12 0
31 689.58 371.64 340.04 1302.96 9.738 1.330 106.280 111.909 2 12 0
32 717.64 389.86 367.08 1219.40 9.938 1.330 107.711 114.122 2 12 0
33 719.69 396.75 360.53 1273.95 5.264 1.330 131.364 137.835 4 14 0
34 687.62 331.97 351.82 1665.79 6.335 1.330 121.395 127.642 4 12 0
35 739.69 402.95 391.02 1292.81 6.090 1.330 120.894 127.001 4 12 0
36 767.75 426.16 415.08 1864.06 6.278 1.330 119.904 126.080 4 12 0
37 785.67 450.17 427.26 2752.28 10.008 1.330 115.488 121.547 4 12 0
38 633.48 288.88 288.34 1311.57 10.410 1.330 128.700 137.113 4 12 1
39 661.53 340.69 312.80 1385.26 10.533 1.331 120.313 128.774 2 12 0
40 689.58 339.05 338.05 1399.50 10.027 1.330 118.948 127.480 2 12 0
41 715.67 424.16 370.92 1258.04 9.734 1.334 121.170 128.906 4 12 0
130
S10
42 661.53 337.39 311.20 1426.34 9.210 1.326 126.238 132.477 4 12 1
43 689.58 359.24 335.02 1431.79 9.273 1.326 118.219 125.579 2 12 0
44 743.72 426.20 396.18 1425.48 12.598 1.331 112.826 120.966 4 12 0
45 689.58 361.41 334.48 1457.78 8.237 1.324 119.234 130.934 4 12 1
46 717.78 387.67 359.18 1613.85 8.780 1.324 111.339 120.691 2 12 0
47 771.78 495.12 420.93 1987.67 9.252 1.324 117.298 124.778 4 12 0
48 537.35 227.62 219.09 1063.21 6.278 1.331 108.407 113.363 4 11 1
49 621.51 343.68 296.51 1549.29 8.507 1.331 98.825 104.036 2 11 0
50a 671.62 341.22 345.91 1233.02 6.170 1.330 112.372 117.504 4 11 0
50b 671.62 362.46 340.48 1207.20 5.614 1.330 108.068 114.896 4 11 0
51 631.46 288.83 286.22 1354.69 12.809 1.318 153.353 164.242 4 12 1
52 687.57 355.00 337.82 1235.64 15.747 1.317 126.985 136.235 2 12 0
53 713.65 391.69 373.29 1457.55 11.945 1.317 137.753 146.294 4 12 0
131
S11
Table S3. Calculated descriptors for the investigated compounds (all values are in eV): energies
of HOMO (EHOMO), LUMO (ELUMO) and their gap (H/L gap)), vertical ionization energy (Ei) in
vacuum and in water (Eis), vertical electron affinity in vacuum (Eea) and in water (Eeas), adiabatic
electron affinity in water (Eeas’). The coloring is based on the subtypes, exerting the same
equatorial ligands (Figure 2).
complex EHOMO ELUMO H/L gap Ei Eis Eea Eeas Eeas'
1 -10.005 -0.487 9.517 9.805 8.400 1.025 2.938 4.225
2a -9.977 -0.447 9.530 9.740 8.279 0.989 2.944 3.976
2b -10.086 -0.608 9.477 9.899 8.402 1.118 2.976 3.988
2c -9.670 -0.109 9.561 9.397 8.348 0.716 2.858 4.102
2d -9.680 -0.133 9.547 9.435 8.216 0.670 2.864 3.898
3 -9.702 -0.331 9.372 9.494 7.428 0.897 2.879 3.915
4 -9.592 -0.316 9.275 9.346 7.299 0.872 2.785 4.124
5 -9.477 -0.249 9.228 9.138 7.211 0.830 2.884 3.920
6 -9.796 -0.465 9.330 9.548 8.243 1.023 2.989 3.841
7 -9.827 -0.504 9.323 9.577 8.325 1.093 2.968 3.997
8 -9.813 -0.491 9.323 9.562 8.252 1.084 2.965 3.996
9 -9.812 -0.489 9.323 9.558 8.253 1.084 2.965 3.995
10 -9.810 -0.486 9.323 9.555 8.251 1.083 2.964 3.997
11 -9.513 -0.420 9.093 9.287 7.323 1.016 2.951 3.982
12 -9.485 -0.430 9.055 9.250 7.277 1.030 2.953 3.986
13 -9.408 -0.421 8.987 9.074 7.184 1.023 2.950 3.981
14 -9.930 -0.573 9.357 9.674 8.228 1.143 2.954 4.060
15 -9.887 -0.524 9.363 9.622 8.224 1.103 2.943 3.971
16 -9.379 -0.512 8.867 9.166 7.236 1.092 2.933 4.058
132
S12
17 -8.932 -1.103 7.829 8.564 7.157 1.781 3.056 4.281
18 -8.596 -0.916 7.680 8.187 7.043 1.583 3.035 4.137
19 -9.880 -0.518 9.362 9.608 8.225 1.107 2.942 3.967
20 -9.874 -0.512 9.362 9.598 8.225 1.104 2.941 3.965
21 -9.425 -0.657 8.769 9.088 7.672 1.265 2.969 3.976
22 -9.219 -0.479 8.741 8.857 7.659 1.096 2.961 3.965
23 -9.210 -0.492 8.719 8.839 7.650 1.112 2.969 3.980
24 -9.210 -0.494 8.716 8.834 7.650 1.117 2.972 3.975
25 -9.200 -0.488 8.712 8.820 7.644 1.113 2.971 3.976
26 -9.104 -0.401 8.703 8.730 7.607 1.034 2.946 3.949
27 -9.510 -0.518 8.992 9.237 7.988 1.305 2.648 4.170
28 -9.431 -0.438 8.992 9.153 7.979 1.234 2.632 4.142
29 -9.421 -0.422 8.999 9.140 7.979 1.224 2.630 4.142
30 -9.419 -0.417 9.002 9.135 7.981 1.223 2.629 4.143
31 -9.410 -0.405 9.004 9.126 7.977 1.211 2.625 4.140
32 -9.415 -0.413 9.002 9.130 7.980 1.222 2.629 4.142
33 -9.453 -0.396 9.057 9.167 7.959 1.205 2.600 4.122
34 -9.410 -0.348 9.061 9.124 7.955 1.159 2.595 4.115
35 -9.400 -0.340 9.060 9.111 7.954 1.153 2.595 4.115
36 -9.390 -0.333 9.057 9.099 7.954 1.148 2.597 4.118
37 -9.409 -0.446 8.963 9.120 7.044 1.262 2.637 4.148
38 -9.430 -0.443 8.986 9.153 7.959 1.244 2.576 4.091
39 -9.413 -0.399 9.014 9.128 7.952 1.208 2.562 4.081
40 -9.412 -0.396 9.016 9.123 7.962 1.210 2.578 4.093
41 -9.354 -0.333 9.021 9.050 7.301 1.145 2.531 4.125
42 -9.455 -0.441 9.014 9.167 7.968 1.262 2.571 4.100
133
S13
43 -9.446 -0.405 9.041 9.150 7.962 1.236 2.561 4.076
44 -8.995 -0.500 8.494 8.598 7.168 1.325 2.616 4.099
45 -9.400 -0.374 9.026 9.107 7.968 1.216 2.587 4.098
46 -9.358 -0.318 9.040 9.062 7.963 1.168 2.579 4.128
47 -9.283 -0.209 9.074 8.979 7.947 1.066 2.545 4.052
48 -9.212 -0.089 9.123 8.877 7.136 0.801 2.351 3.872
49 -9.166 0.010 9.176 8.793 7.133 0.722 2.334 3.838
50a -9.133 0.088 9.400 8.747 7.116 0.649 2.303 3.806
50b -9.312 -0.092 9.041 8.887 7.148 0.849 2.358 3.898
51 -9.327 -0.480 8.847 9.033 7.878 1.290 2.735 4.094
52 -9.130 -0.277 8.853 8.828 7.870 1.100 2.718 4.076
53 -9.109 -0.213 8.896 8.802 7.858 1.044 2.692 4.056
134
S14
Table S4. QSAR models for the cytotoxicity in the cell line CH1 with simulated annealing
chosen combination of descriptors. (Q2, AAR´and RMS´ are the R2, AAR and RMS values for
cross validated predictions, using LOOP; the derived coefficients are for a model equation with
auto scaled values of the descriptors).
Model Number of variables
Used descriptors R2 Q2 AAR AAR' RMS RMS' Coefficients
1 1 q(Pt) 0.4132 0.3920 0.6318 0.6431 0.8241 0.8389 -0.643
2 1 Hacc 0.5130 0.4953 0.5695 0.5795 0.7508 0.7643 -0.716
3 2 Hdon, Hacc 0.6978 0.6769 0.4707 0.4878 0.5914 0.6115 -0.435, -0.651
4 2 q(Pt), Hdon 0.6847 0.6643 0.4698 0.4843 0.6041 0.6233 -0.633, -0.522
5 3 Hdon, Hacc,
COOH 0.7450 0.7226 0.4160 0.4355 0.5433 0.5666 -0.363, -0.647, -0.229
6 3 q(Pt), Eeas
’, Hdon
0.7867 0.7641 0.3794 0.4000 0.4968 0.5226 -0.516, -0.340, -0.513
7 3 q(Pt), Hdon,
COOH 0.7130 0.6866 0.4415 0.4620 0.5763 0.6023 -0.617, -0.466, -0.178
8 3 MW, q(Pt), Hdon
0.7122 0.6800 0.4271 0.4505 0.5769 0.6087 0.187, -0.716, -0.541
9 3 �, q(Pt), Hdon
0.7285 0.6987 0.4088 0.4306 0.5603 0.5905 0.224, -0.710, -0.546
10 3 q(Pt), dEis, Hdon
0.7219 0.6910 0.4305 0.4529 0.5674 0.5980 -0.652, -0.227, -0.639
11 3 Eeas’, Hdon, Hacc
0.7768 0.7542 0.3998 0.4210 0.5083 0.5334 -0.308, -0.444,
135
S15
-0.525
12 4 MW, Eeas
’, Hdon, Hacc
0.8459 0.8228 0.3434 0.3695 0.4223 0.4529
0.309, -0.359, -0.456, -0.656
13 4 MW, q(Pt), Eeas
’, Hdon 0.8458 0.8196 0.3324 0.3595 0.4225 0.4570
0.280, -0.621, -0.399, -0.539
14 4 q(Pt), Ei,
Eeas’, Hdon
0.8137 0.7802 0.3611 0.3921 0.4643 0.5044
-0.584, -0.180, -0.336, -0.543
15 5 MW, �,
q(Pt), Eeas’,
Hdon 0.8713 0.8471 0.3178 0.3484 0.3859 0.4207
-1.056, 1.268, -0.487, -0.316, -0.541
16 5 �, Eea, Eeas
’, Hdon, Hacc
0.8505 0.8229 0.3393 0.3721 0.4160 0.4527
0.302, -0.051, -0.309, -0.462, -0.625
136
S16
Table S5. External validation of the best models for cytotoxicity in CH1 cells, obtained with three, four or five descriptors:
Model N
Training set
Number of variables
Used descriptors R2 Q2
Pred R2
pred AAR
pred RMS
a 0.751 0.7197 0.7210 0.402 0.5394
b 0.7278 0.6879 0.7828 0.5039 0.7049
c 0.7728 0.7383 0.6263 0.5655 0.6976
d 0.7239 0.6924 0.808 0.3738 0.4831
5
e
3 Hdon, Hacc, COOH
0.7532 0.7263 0.7139 0.4734 0.5492
a 0.7931 0.7652 0.7560 0.3726 0.5044
b 0.7699 0.7289 0.8158 0.4782 0.6492
c 0.8085 0.7731 0.7043 0.4672 0.6206
d 0.7778 0.7492 0.8471 0.3511 0.4311
6
e
3 q(Pt),
Eeas', Hdon
0.7868 0.7622 0.7794 0.3765 0.4822
a 0.7329 0.6872 0.6390 0.4236 0.6136
b 0.6382 0.5864 0.7908 0.4658 0.692
c 0.7249 0.6782 0.5843 0.6199 0.7358
d 0.6898 0.6414 0.7452 0.4671 0.5565
8
e
3 MW, q(Pt), Hdon
0.8358 0.8132 0.0635 0.7271 0.9935
a 0.748 0.7056 0.6593 0.4084 0.596
b 0.6512 0.6012 0.8116 0.443 0.6566
c 0.7445 0.7029 0.5970 0.6029 0.7245
d 0.7052 0.6587 0.7662 0.4502 0.5332
9
e
3 �, q(Pt),
Hdon
0.8395 0.8176 0.1942 0.6843 0.9216
a 0.7804 0.7511 0.7536 0.3976 0.5068
b 0.7606 0.7191 0.8103 0.4987 0.6589
11
c
3 Eeas', Hdon, Hacc
0.8018 0.7705 0.7116 0.4759 0.6129
137
S17
d 0.7652 0.7368 0.8474 0.3648 0.4307
e 0.7649 0.7363 0.8346 0.3534 0.4176
a 0.8614 0.8313 0.7753 0.4361 0.484
b 0.8381 0.8027 0.8546 0.434 0.5767
c 0.8736 0.8478 0.754 0.4838 0.566
d 0.8303 0.7966 0.9141 0.2398 0.3232
12
e
4 MW, Eeas', Hdon, Hacc
0.8459 0.8112 0.842 0.3824 0.4082
a 0.8553 0.8199 0.8023 0.3662 0.454
b 0.8422 0.8054 0.8385 0.4723 0.608
c 0.8644 0.8285 0.7494 0.4912 0.5713
d 0.8297 0.7913 0.9199 0.2564 0.3119
13
e
4 MW, q(Pt),
Eeas', Hdon
0.8729 0.8435 0.6236 0.5156 0.6299
a 0.8275 0.773 0.7422 0.4167 0.5185
b 0.7723 0.7215 0.8454 0.4307 0.5949
c 0.8524 0.8155 0.6043 0.598 0.7179
d 0.8176 0.7766 0.8157 0.3818 0.4734
14
e
4 q(Pt), Ei, Eeas', Hdon
0.8163 0.774 0.8161 0.3614 0.4403
a 0.8646 0.8154 0.7701 0.445 0.4896
b 0.8486 0.8041 0.8365 0.4554 0.6117
c 0.8785 0.8416 0.7246 0.5217 0.5989
d 0.8352 0.7956 0.9176 0.2341 0.3165
15
e
5 �, Eea,
Eeas', Hdon, Hacc
0.8494 0.8105 0.8491 0.3615 0.3989
a 0.8769 0.8438 0.8439 0.34 0.4034
b 0.8648 0.8296 0.8802 0.4132 0.5236
c 0.8894 0.857 0.8073 0.4262 0.501
d 0.8574 0.8216 0.9352 0.2446 0.2806
16
e
5 MW, �, q(Pt),
Eeas', Hdon
0.8787 0.8455 0.7716 0.4136 0.4906
138
S18
Table S6. QSAR models for cytotoxicity in the cell line SW480 with simulated annealing
chosen combination of descriptors (Q2, AAR´and RMS´ are the R2, AAR and RMS values for
cross validated predictions, using LOOP; the derived coefficients are for a model equation with
auto scaled values of the descriptors).
Model Number
of variables
Used descriptors R2 Q2 AAR AAR' RMS RMS' Coefficients
1 1 q(Pt) 0.5453 0.5292 0.5847 0.5950 0.7388 0.7518 -0.738
2 2 Hdon, Hacc 0.7009 0.6785 0.4446 0.4620 0.5992 0.6212 -0.321, -0.727
3 2 q(Pt), Hdon 0.7196 0.7010 0.4173 0.4315 0.5802 0.5991 -0.731, -0.418
4 3 q(Pt), Es’,
Hdon 0.7535 0.7247 0.4246 0.4495 0.5440 0.5749
-0.813, -0.220, -0.505
5 3 q(Pt), Ei,
Hdon 0.7587 0.7259 0.4083 0.4337 0.5382 0.5736
-0.811, -0.217, -0.454
6 3 q(Pt), EHOMO, Hdon
0.7552 0.7213 0.4121 0.4381 0.5422 0.5784 -0.812, 0.210, -0.461
7 3 �, q(Pt), Hdon
0.7466 0.7208 0.4312 0.4539 0.5515 0.5789 0.176, -0.791, -0.436
8 3 Es’, Hdon, Hacc
0.7607 0.7282 0.4151 0.4419 0.5360 0.5712 -0.304, -0.423, -0.861
9 4 Es, Hdon, Hacc, COOH
0.7893 0.7522 0.4084 0.4426 0.5030 0.5454
-0.334, -0.387, -0.867, -0.170
10 4 Es’, Hdon, 0.7875 0.7507 0.4097 0.4438 0.5050 0.5470 -0.329, -0.377,
139
S19
Hacc, COOH -0.869, -0.174
11 4 q(Pt),
ELUMO, Ei, Hdon
0.7811 0.7486 0.4084 0.4395 0.5126 0.5494
-0.891, 0.167, -0.244, -0.457
12 4 Ei, Eea, Eeas,
Hdon 0.8094 0.7834 0.3891 0.4167 0.4783 0.5099
-0.307, -0.412, 0.845, -0.431
13 4 Ei, Eeas,
Eeas’, Hdon 0.7965 0.7648 0.3900 0.4197 0.4943 0.5314
-0.185, 0.711, -0.365, -0.412
14 5 Ei, Eea, Es, Eeas, Hdon
0.8230 0.7912 0.3784 0.4141 0.4610 0.5007
-0.281, -0.459, -0.147, 0.871, -0.486
15 5 q(Pt),
H/Lgap, Ei, Eeas, Hdon
0.8145 0.7768 0.3798 0.4157 0.4719 0.5176
-0.329, 0.678, -0.818, 0.695, -0.409
140
S20
Table S7. External validation of the best models for SW480 cells, obtained with three, four or
five descriptors:
Model N
Training set
Number of variables
Used descriptors
R2 Q2 Pred R2 pred AAR
pred RMS
a 0.7655 0.7280 0.6834 0.4844 0.6029
b 0.6899 0.6304 0.7981 0.6115 0.7160
c 0.7905 0.7440 0.4651 0.7380 0.8212
d 0.7467 0.7087 0.7074 0.5710 0.7321
4
e
3 q(Pt), Es´,
Hdon
0.8262 0.8028 0.3439 0.7227 0.8329
a 0.7519 0.7042 0.7481 0.4249 0.5379
b 0.6644 0.6111 0.8211 0.5277 0.6740
c 0.8147 0.7812 0.3419 0.7643 0.9109
d 0.7409 0.6936 0.7483 0.4843 0.6790
6
e
3 q(Pt),
EHOMO, Hdon
0.8288 0.7975 0.3705 0.6516 0.8159
a 0.7915 0.7538 0.6227 0.5458 0.6582
b 0.7294 0.6824 0.7667 0.6592 0.7697
c 0.8040 0.7485 0.5766 0.6131 0.7307
d 0.7385 0.6908 0.7976 0.4893 0.6088
8
e
3 Es, Hdon,
Hacc
0.7836 0.7450 0.6809 0.4268 0.5809
a 0.8119 0.7644 0.6638 0.5392 0.6213
b 0.7482 0.6909 0.7892 0.6224 0.7316
c 0.8270 0.7681 0.5750 0.6405 0.7320
d 0.7843 0.7340 0.7593 0.5677 0.6640
9
e
4 Es, Hdon,
Hacc, COOH
0.8241 0.7848 0.5864 0.5129 0.6613
11 a 4 q(Pt), 0.7684 0.6919 0.8088 0.4027 0.4686
141
S21
b 0.7034 0.6534 0.8608 0.5016 0.5945
c 0.8178 0.7769 0.4493 0.7042 0.8333
d 0.7571 0.7048 0.8254 0.4502 0.5656
e
ELUMO, Ei, Hdon
0.8439 0.8118 0.5137 0.5467 0.7171
a 0.8045 0.7716 0.8003 0.4235 0.4789
b 0.7451 0.6966 0.8799 0.4801 0.5522
c 0.8531 0.8138 0.5918 0.6445 0.7174
d 0.8033 0.7667 0.7904 0.4678 0.6196
12
e
4 Ei, Eea,
Eeas, Hdon
0.8409 0.8116 0.4570 0.6464 0.7578
a 0.8009 0.7550 0.7445 0.4786 0.5416
b 0.7312 0.6669 0.8705 0.4677 0.5734
c 0.8577 0.8208 0.5174 0.6361 0.7801
d 0.7834 0.7379 0.7856 0.4471 0.6266
13
e
4 Ei, Eeas, Eeas’, Hdon
0.8195 0.7797 0.5191 0.6080 0.7131
a 0.8274 0.7486 0.7697 0.4388 0.5142
b 0.7466 0.6722 0.9020 0.4415 0.4988
c 0.8761 0.8364 0.5470 0.6869 0.7557
d 0.8222 0.7824 0.7861 0.4741 0.6260
14
e
5 Ei, Eea, Es, Eeas, Hdon
0.8527 0.8251 0.4405 0.6629 0.7692
a 0.8057 0.7410 0.8381 0.3838 0.4311
b 0.7577 0.6872 0.8924 0.4661 0.5228
c 0.8554 0.8006 0.6248 0.6118 0.6878
d 0.8019 0.7461 0.8268 0.4338 0.5632
15
e
5
q(Pt), H/Lgap, Ei, Eeas,
Hdon
0.8516 0.8181 0.5809 0.5569 0.6657
142
S22
Table S8. Statistical parameters for the best four-variable models, using IC50 = 600, 1000 or
2000 μM as input for the inactive compounds from subset 2.
model 1 model 2 model 3
input IC50 R2 Q2(LOOP) AAR R2 Q2(LOOP) AAR R2 Q2(LOOP) AAR
600 0.79 0.75 0.41 0.80 0.77 0.39 0.81 0.78 0.39
1000 0.80 0.76 0.43 0.80 0.77 0.40 0.82 0.80 0.40
2000 0.80 0.77 0.47 0.81 0.78 0.43 0.83 0.80 0.42
Table S9. Elemental analysis data.
Compound Formula MW Calculated (%) Found (%)
C H N C H N
nedaplatin C2H8N2O3Pt 303.17 7.92 2.66 9.24 8.03 2.45 9.05
48 C10H18N2O11Pt 537.33 22.35 3.38 5.21 22.32 3.24 5.04
49 C16H30N2O11Pt·0.5H2O 630.50 30.48 4.96 4.44 30.41 4.59 4.39
50 C20H36N4O9Pt·0.5H2O 680.61 35.29 5.48 8.23 35.15 5.10 7.98
143
S23
Table S10. Crystal data and details of data collection for 7
Complex 7
empirical formula C12H22Cl2N2O8Pt
fw 588.31
space group P-1
a, Å 8.2185(3)
b, Å 10.0375(4)
c, Å 12.4165(5)
V, Å3 891.63(6)
Z 2
�, Å 0.71073
calcd, g cm-3 2.191
crystal size, mm 0.10 x 0.08 x 0.02
T, K 100
�, mm-1 8.211
R1a 0.0234
wR2b 0.0486
GOFc 0.998
a R1 = ||Fo| - |Fc||/|Fo|, b wR2 = { [w (Fo
2 - Fc2)2] /[w(Fo2)2 ]}1/2. c GOF = {[w(Fo2 -
Fc2)2] /(n –p)}1/2, where n is the number of reflections and p is the total number of parameters
refined.
144
III. CONCLUSIONS
In this PhD thesis, the synthesis, characterization, pharmacological, theoretical and
QSAR investigations of 29 novel bis-, tris- and tetrakis(carboxylato)platinum(IV)
complexes with potential antitumor activity are reported.
In the first part of the work, a series of six novel
bis(carboxylato)dichloridobis(ethylamine)platinum(IV) complexes, featuring different
axial ligands have been synthesized and fully characterized. Their lipophilicity (as log
Po/w values) and cytotoxicity in four human tumor cell lines were evaluated and structure-
activity relationships were drawn. The most active complexes demonstrated IC50 values
in the low nanomolar range, significantly lower than that of cisplatin in both, cisplatin
sensitive and resistant cell lines. Linear correlation between lipophilicity and cytotoxicity
in the tested cell lines was observed in the series, while the most lipophilic compounds
were the most cytotoxic. However, the complex, featuring an axial cyclopentylamide
residue did not follow this trend, as a higher cytotoxicity could be expected from its log
Po/w value.
In the second part, 21 new tetracarboxylatoplatinum(IV) complexes, designed as
prodrugs of carboplatin were synthesized, characterized and their lipohilicity,
cytotoxicitiy and redox potentials were determined. Contrary to corresponding
diam(m)inebis(carboxylato)dichloridoplatinum(IV) compounds, cytotoxicity could not
significantly be enhanced by variation of the axial ligands and their lipophilicity. Even
the most lipophilic complexes possessed lower cytotoxic potency than carboplatin. In
addition, the rate of reduction by ascorbic acid of a chosen
diamminetetrakis(carboxylato)platinum(IV) compound and its diamine-
bis(carboxylato)dichlorido analogue, featuring the same axial ligands were examined.
145
The considerable difference found between the redox kinetics is in accordance with large
variance in cytotoxicity, but not with similar redox potentials.
In the last part of the work, theoretical and QSAR investigations on 53
platinum(IV) complexes, bearing the equatorial sphere of cisplatin, its bis(ethylamine)
analogue, its ethylendiamine analogue, carboplatin, nedaplatin and oxaliplatin were
performed. The compounds used for the study were synthesized, characterized and tested
for cytotoxicity in our laboratories, more than halve of them within this PhD work. The
hybrid DFT functional wb97x was utilized for geometry structure optimization and
calculation of several molecular properties of the complexes. On basis of the
computations, the compounds were divided in subsets and an attempt for explanation of
their divergent physicochemical properties was made. Furthermore, reliable, robust and
predictive models for their cytotoxic activity in CH1 and SW480 cells were developed.
To the best of our knowledge, this was the first report of DFT descriptors-based QSAR
models for the in vitro cytotoxicity of platinum(IV) complexes.
The influence of the type of axial and equatorial ligands on the cytotoxic potency of
platinum(IV) complexes was studied by means of analytical and theoretical methods. For
this purpose, appropriate procedures for the preparation of various platinum(IV)
compounds, prodrugs of clinically applied cytostatics and their analogues were
developed. The obtained results represent one further step towards a better understanding
of the biological behavior of platinum(IV) carboxylato complexes and their subsequent
rational development. However, there are still many open questions, concerning the fate
of platinum(IV)-based drugs in vivo and the ways to control their extracellular and
intracellular pharmacokinetics. The ongoing in vivo studies with chosen compounds from
146
the series, developed within this PhD work, together with further analytical and
theoretical investigations are expected to contribute to a better fine-tuning of successful
platinum(IV)-based chemotherapeutics.
147
148
Curriculum vitae
Personal information
Surname(s) / First name(s) Varbanov, Hristo Plamenov
Address(es) Starkfriedgasse 15 / 3205, 1180 Vienna, Austria
Telephone(s) Mobile +4369917123262
E-mail(s) [email protected]; [email protected]
Nationality Bulgarian
Date and Place of birth 12th February 1984; Sofia, Bulgaria
Gender Male
Marital status Single
Education and training
Dates 15th February 2009 – to date
PhD Research Novel antineoplastic platinum(IV) complexes: Synthesis, characterization, biological
investigations and structure-activity relationships
Name and type of organisation
providing education and training
Bioinorganic Chemistry group (M. Galanski, B.K. Keppler), Department of Inorganic
Chemistry, University of Vienna, Vienna, Austria
Dates 05th September – 01st December 2011
Research work DFT calculations of anticancer Pt(IV) complexes; building of QSAR and QSPR models.
Name and type of organisation
providing education and training
Biomodelling group (F. Jensen), Department of Chemistry, Aarhus University, Aarhus,
Denmark
149
Dates September 2003 – December 2008
Title of qualification awarded Magister of Pharmacy
Diploma thesis Synthesis, chemometric and pharmacological investigation of novel platinum and
palladium complexes with 5-methyl-5-(4-pyridil)hydantoin and its derivatives.
Name and type of organisation
providing education and training Faculty of Pharmacy, Medical University of Sofia, Sofia, Bulgaria
Dates March 2005 – December 2008
Research work Synthesis and characterization of new platinum and palladium complexes with hydantoin
derivatives.
Name and type of organisation
providing education and training
Department of Chemistry (A. Bakalova), Faculty of Pharmacy, Medical University of
Sofia, Sofia, Bulgaria
Dates 05th June 2007 – 28th August 2007
Research work: Chiral separation of aminoacids and hydantoin derivatives, using HPLC and micro HPLC
techniques
Name and type of organisation
providing education and training
Drug analysis group (G. Gübitz, M. Schmid), Department of Pharmaceutical Chemistry,
Karl-Franzens-University Graz, Graz, Austria
Dates February 2006 – February 2008
Specialization Industrial Pharmacy
Name and type of organisation
providing education and training Faculty of Pharmacy, Medical University - Sofia, Sofia, Bulgaria
Dates September 1998 - May 2003
Title of qualification awarded Diploma of Completed Secondary Education
Name and type of organisation
providing education and training
Chemistry class in the National High School of Mathematics and Science ‘acad. L.
Chakalov’, Sofia, Bulgaria
Dates September 1991 - June 1998
Title of qualification awarded Basic Education Completion Certificate
Name and type of organisation
providing education and training 131st primary school ‘K.A. Timiriazev’, Sofia, Bulgaria
150
Language skills
Mother tongue Bulgarian
Other language(s)
Self-assessment Understanding Speaking Writing
Listening Reading Spoken interaction Spoken production
English C1 C1 C1 C1 C1
German B2 B2 B2 B2 B2
Russian B1 B1 B1 A2 A2
Professional skills and
research interests
Synthesis and characterisation (using 1D and 2D NMR techniques, IR spectroscopy, MS,
HPLC, TG, X- RAY, DFT methods) of metal complexes with biological activity
Theoretical, QSAR and QSPR studies of bioactive metal complexes
Analytics and bioanalytics
Publications in scientific journals
Theoretical Investigations and Density Functional Theory Based Quantitative Structure–Activity Relationships Model for Novel Cytotoxic Platinum(IV) Complexes. H.P. Varbanov, M.A. Jakupec, A. Roller, F. Jensen, M. Galanski, B.K. Keppler; J. Med. Chem., 2013, 56, 330-344.
Novel tetracarboxylatoplatinum(IV) complexes as carboplatin prodrugs. H.P. Varbanov, S.M. Valiahdi, C.R. Kowol, M.A. Jakupec, M. Galanski, B.K. Keppler; Dalton Trans., 2012, 41, 14404-14415. (with cover paper).
Synthesis and characterization of novel bis(carboxylato)dichloridobis(ethylamine) platinum(IV) complexes with higher cytotoxicity than cisplatin. H. Varbanov, S.M. Valiahdi, A.A. Legin, M.A. Jakupec, A. Roller, M. Galanski, B.K. Keppler; Eur. J. Med. Chem., 2011, 46, 5456-5464.
Synthesis of palladium(II) complexes with 3-amino-5-methyl-5-(4-pyridyl)-hydantoin: cytotoxic and antimicrobial investigations and comparison with their platinum analogues. H. Varbanov, R. Buyukliev, A. Bakalova, G. Momekov, R. Baykushev; Trans. Met. Chem., 2010, 35, 457–461
Novel Pt(II) and Pt(IV) complexes with 3-amino-5-methyl-5-(4-pyridyl)- 2,4-imidazolidenedione. Synthesis, physicochemical, chemometric and pharmacological investigation. A. Bakalova, H. Varbanov, R. Buyukliev, S. Stanchev, G. Momekov, D. Ivanov; Inorg. Chim. Acta, 2010, 363, 1568–1576.
151
3-Amino-5-methyl-5-(4-pyridyl)hydantoin. H. Varbanov, R. Buyukliev, A. Bakalova, A. Roller; Acta Cryst., 2009, E65, o953.
In Vitro Biochemical and Pharmacological Evaluation of a Novel Cytotoxic Dinuclear Pt(II) Complex with 3-amino-5-methyl-5-phenylhydantoin: Cytotoxicity, Induction of Apoptosis, DNA-binding and Processing of the DNA Adducts. G. T. Momekov, I. Ugrinova, E. A. Pasheva, A. G. Bakalova, H. P. Varbanov, D. V. Ferdinandov, D. S. Ivanov,S. M. Konstantinov; Annals of the New York Academy of Sciences, 2009, 1171, 649–658.
DFT study of the structure and spectral behavior of new Pt(II) complexes with 5-methyl-5(4-pyridyl)hydantoin. A. Bakalova, H. Varbanov, S. Stanchev, D. Ivanov, F. Jensen; Int. J. Quant. Chem., 2009, 109, 826-836.
Palladium(II) complexes with 5-methyl-5-(4-pyridil)-2,4-imidazolidendione. Synthesis, thermogravimetric and cytotoxic investigation. A. Bakalova, H. Varbanov, R. Buyukliev, G. Momekov, D. Ivanov; J. Therm. Anal.and Cal., 2009, 95, 241-246.
Synthesis, characterization and biological activity of Pt(II) and Pt(IV) complexes with 5-methyl-5-(4-pyridyl)-2,4-imidazolidenedione. A. Bakalova, H. Varbanov, R. Buyukliev, G. Momekov, D. Ferdinandov, S. Konstantinov and D. Ivanov; Eur. J. Med. Chem., 2008, 43, 958-965.
Synthesis and crystal structure of new Pt(II) complex with 3-amino-5-methyl-5-phenyl hydantoin.A. Bakalova, R. Petrova, B. Shivatchev, H. Varbanov; J. Coord. Chem., 2007, 60, 15, 1701-1707.
Participation in conferences, workshops and summer schools:
XIth International Symposium on Platinum Coordination Compounds in Cancer Chemotherapy (ISPCC XI), October 2012 in Verona, Italy – oral presentation (in English): ‘Pt(IV) bis-, tris- and tetracarboxylato complexes as potential anticancer drugs: synthesis, analytical, biological, DFT and QSAR studies’
11th European Biological Inorganic Chemistry Conference (EUROBIC 11), September 2012, Granada, Spain – poster presentation: ‘Theoretical investigations and a DFT based QSAR model for novel cytotoxic Pt(IV) complexes’
7th Workshop on Inorganic Chemistry in Austria 2012 (WACÖ 2012), April 2012 in Innsbruck, Austria – oral presentation (in English): ‘Novel tetracarboxylato platinum(IV) complexes as carboplatin prodrugs’
15th International conference on Bioinorganic Chemistry (ICBIC 15), August 2011 in Vancouver, Canada – poster presentation: ‘Novel tetra- and tricarboxylato platinum(IV) complexes as carboplatin and nedaplatin prodrugs: synthesis, cytotoxicity and SAR’
10th European Bioinorganic Chemistry Conference (EUROBIC 10), June 2010 in Thessaloniki, Greece – poster presentation: ‘Novel platinum(IV) complexes with high cytotoxicity’
6th Workshop on Inorganic Chemistry in Austria 2010 (WACÖ 2010), March 2010 in Linz, Austria – oral presentation (in English): ‘Towards the development of novel anticancer platinum(IV) complexes’
Student Science Session of Pharmacy, November 2008 in Sofia, Bulgaria – oral presentation (in Bulgarian): ‘Synthesis, chemometric and pharamacological investigation of new platinum and palladium complexes with 5-methyl-5(4-pyridyl)hydantoin and its derivatives’
38th International Conferences on Coordination Chemistry, July 2008 in Jerusalem, Israel - poster presentation: ‘Theoretical and spectroscopic study of new platinum(II) complexes with 5-methyl-5(4-pyridyl)-2,4-imidazolidenedione’ and co-author in poster: ‘Comparative physicochemical and pharmacological investigation of new Platinum(II) and Palladium(II) complexes with 5-methyl-5(4-pyridyl)hydantoin’
152
8th International Symposium and Summer school on Bioanalysis, June 2008 in Nitra, Slovakia - poster presentation: ‘Enantioseparation of new amino acid analogues and hydantoin derivatives using chiral HPLC techniques’
7th National Conference of chemistry for diploma and PhD students May 2008 in Sofia, Bulgaria - oral presentation (in Bulgarian): ‘Theoretical, Spectroscopic and pharmacological investigation of new platinum complexes with 5-methyl-5(4-pyridyl)hydantoin’
Student Science Session of Pharmacy, November 2007 in Sofia, Bulgaria – oral presentation (in Bulgarian): ‘Synthesis, characterisation and pharmacological study of new platinum complexes with 5-methyl-5(4-pyridyl)hydantoin’
13th International Conference on Biological Inorganic Chemistry, July 2007 in Vienna, Austria - poster presentation: ‘New Pt(II) and Pt(IV) complexes with 5-methyl-5(4-pyridyl)-2,4-imidazolidenedione and different inorganic ligands. Synthesis, characterization and cytotoxic activity’
7th International Symposium and summer school on Bioanalysis, June 2007 in Pec, Hungary
37th International Conferences on Coordination Chemistry, August 2006 in CapeTown, South Africa – poster presentation: ‘Synthesis and crystal structure of new Pt(II) complexes with some hydantoin derivatives’
Granted projects
Student research project ‘Synthesis and physicochemical characterization of new platinum and palladium complexes with hydantoin derivatives and different inorganic ions. Pharmacological investigation for cytotoxic activity in vitro of the new compounds’ from the Student Union of Medical University-Sofia, approved in 2007
Awards
Scholarship for excellence in chemistry from the foundation ‘Evrika’ on the name of acad. Rostislav Kaishev for the years 2005, 2006 and 2007
First prize in the competition ‘Shimadzu’ of the ‘Union of Chemists in Bulgaria’ for best diploma work for 2009 year
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